Methods and formulations comprising agonists and antagonists of nuclear hormone receptors

ABSTRACT

Novel compounds, pharmaceutical compositions, and methods are provided for modulating processes mediated by nuclear hormone receptors. A partial or complete agonist or antagonist modulates, directly or indirectly, an activity of one or more nuclear hormone receptors for glucocorticoids (GRs), androgens (ARs), mineralocorticoids (MRs), progestins (PRs), estrogens (ERs), thyroid hormones (TRs), vitamin D (VDRs), retinoids (RARs and RXRs), peroxisomes (XPARs and PPARs), icosanoids (IRs), or one or more orphan receptors, such as steroid and thyroid receptors. Exemplary compounds of the disclosure are bacterial products, for example bacterial toxins, and these compounds are useful in screens for other antagonists and agonists. Related methods and compositions are provided for diagnosis, treatment and prevention of bacterial disease and associated or unrelated inflammatory, autoimmune, toxic (including shock), and chronic and/or lethal sequelae associated with bacterial infection.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Application No.60/416,222, filed Oct. 4, 2002, and U.S. Provisional Application No.60/419,454, filed Oct. 18, 2002. Both of these provisional applicationsare incorporated herein in their entirety.

FIELD

This application relates to methods for identifying agonists andantagonists of a nuclear hormone receptor using bacterial products.

BACKGROUND

The effectiveness of known modulators of steroid receptors is oftencompromised by their undesired side-effect profile, particularly afterlong-term administration. For example, the effectiveness of progesteroneand estrogen agonists, such as norgestrel and diethylstilbesterolrespectively, as female birth control agents must be weighed against theincreased risk of breast cancer and heart disease to women taking suchagents. Similarly, the progesterone antagonist, mifepristone (RU486), ifadministered for chronic indications, such as uterine fibroids,endometriosis and certain hormone-dependent cancers, could lead tohomeostatic imbalances in a patient due to its inherent cross-reactivityas a GR antagonist. Accordingly, identification of additional compoundsand methods for modulating activity of nuclear hormone receptors will beof significant value in the treatment of a wide range of diseases.

Although there are compositions and methods proposed in the art formodulating nuclear hormone receptor activity and thereby amelioratingdisease mediated directly or indirectly by the action of nuclear hormonereceptors, there is a continuing need for and a continuing search in thefield for additional and more effective compositions and methods tosatisfy these objectives. Thus, the identification of compounds andmethods that effectively modulate nuclear hormone receptor activity withminimal side effects remains an important objective in the art.

SUMMARY

Compounds, pharmaceutical compositions, and methods for modulatingprocesses mediated by nuclear hormone receptors are provided herein.

In one embodiment, methods are provided for identifying a compound thathas an effect of a partial or complete agonist or antagonist of one ormore nuclear hormone receptors for glucocorticoids (GRs), androgens(ARs), mincralocorticoids (MRs), progestins (PRs), estrogens (ERs),thyroid hormones (TRs), vitamin D (VDRs), retinoids (RARs and RXRs),peroxisomes (XPARs and PPARs), icosanoids (IRs), or one or more orphanreceptors, such as steroid and thyroid receptors.

Methods and pharmaceutical compositions are provided for treatment andprevention of bacterial disease and associated or unrelatedinflammatory, autoimmune, toxic (including shock), and chronic and/orlethal sequelae associated with bacterial infection. In related aspects,methods and pharmaceutical compositions are provided for treatment andprevention inflammatory, autoimmune immunological, lethal and toxicsymptoms and diseases not causally associated with bacterial infection.These methods and compositions generally employ one or more agonists orantagonists of a nuclear hormone receptor as described herein.

Methods are further provided for identifying a compound that has theeffect of an agonist or antagonist of a nuclear hormone receptor. Inexemplary embodiments these methods generally include the steps ofproviding viable cells that express a nuclear hormone receptor and anuclear hormone receptor reporter construct, wherein expression of thesubstrate reporter construct is detectable and provides a measurement ofnuclear hormone receptor pathway activation or repression. Test cellsare contacted with a test agent and a bacterial product, and controlcells are contacted with the bacterial product alone. Then nuclearhormone receptor pathway activation or repression is detected andcompared between the test and control cells to identify a test agentthat modulates activation or repression of the nuclear hormone receptorpathway activity by the bacterial product. Related methods are providedfor identifying cofactors, nuclear hormone receptors and other usefulagents that interact directly or indirectly with bacterial products tomediate activation or repression of nuclear hormone receptors.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are two graphs showing anthrax lethal toxin (LeTx)repression of Dexamethasone-(Dex)-induced glucocorticoid receptor (GR)transactivation in Cos7 cells. Cos7 cells were plated out at a densityof 5×10⁵ cells/well in 24-well plates in Dulbecco's modified Eagle'smedium (DMEM) containing 10% charcoal-stripped serum, 10 μg/1 mlpenicillin-streptomycin and 2 mM glutamine one day prior totransfection. Cos7 cells were transfected overnight with 20 ng SVglucocorticoid receptor (SVGR), 100 ng glucocorticoid responseelement-luciferase reporter construct (GRE-TK luc), 60 ng pSG5(Stratagene) and 20 ng PRL TK (Promega, constitutive renilla luciferasecontrol) using Fugene6 (Roche) according to Manufacturer's instructions.The media was then replaced with DMEM containing 10% charcoal-strippedmedia, 100 nM Dex and either with increasing concentrations of LF, alone(●) or in the presence of 500 ng/ml PA (◯) (FIGS. 1A and 1B); or withincreasing concentrations of E687C, either alone (▪) or in the presenceof 500 ng/ml PA (□) (1B). After 24 hours the cells were lysed andfirefly and renilla luciferase assayed using the dual luciferase assay(Promega). The firefly luciferase activity was normalized to the renillaluciferase to control for differences in cell number and transfectionefficiency. The induction was calculated as the mean of triplicatenormalized luciferase samples in the presence of 100 nM Dex divided bythe mean normalized luciferase in the absence of Dex. In order tocompare separate experiments the induction was set to 100% for 100 nMDex treatment only (for example, no lethal factor (LF) or protectiveantigen (PA)) and the other data points normalized to this accordingly.The data shown is the mean and standard deviation of eight (FIG. 1A) orthree (FIG. 1B) experiments. A one-way analysis of variance (ANOVA)followed by a Dunnett post hoc test was performed between 100 nM Dexonly treatments and 100 nM Dex and LF and/or PA treatments. A singleasterisk (*) designates a p value of 0.01-0.05; a double asterisk (**)designates a p value of 0.001-0.01; a triple asterisk (***) designates ap value of <0.001.

FIGS. 2A and 2B are two graphs showing a comparison of the effects ofmifepristone (RU 486) and LeTx on the dose response curve of Dex inGR-transfected cos7 cells. Cos7 cells were transfected as described forFIG. 1. After transfection, the media was replaced with DMEM containing10% charcoal-stripped media and increasing concentrations of Dex, eitheralone (▪) or in the presence of 0.2 μM RU 486 (□), 500 ng/ml PA and 5ng/ml LF (●) or 50 ng/ml LF and 10 ng/ml PA (◯). After twenty-four hoursthe cells were lysed as described above. The mean and standard deviationof three experiments are shown in FIG. 2A. The renilla normalizedluciferase data (standardized to 100 for 1 μM Dex in each individualexperiment) is shown with the data normalized as a percentage of maximalfor each treatment shown in FIG. 2B (inset).

FIG. 3 is a graph showing a comparison of the effects of LeTx on themutant 407C and wild type GR. Cos7 cells were transfected using eitherthe same plasmid mix as described in FIG. 1 containing the wild type GR(□) or with the mutant 407C GR, which lacks the N-terminaltransactivation domain (◯). After transfection, the media was replacedwith DMEM containing 10% charcoal-stripped media, 1 μM Dex, andincreasing concentrations of the LF in the presence of 500 ng/ml PA.After 24 hours the cells were lysed and firefly luciferase values werenormalized to renilla luciferase. Experiments were then compiled bystandardization as described for FIG. 1. The mean +/− standard deviationof three experiments is shown. Statistics were performed using a one-wayANOVA followed by a Bonferroni post hoc test.

FIG. 4 is a graph showing LeTx repression of dexamethasone inducedtyrosine aminotransferase (TAT) in hepatoma cell line (HTC) cells. HTCcells were plated out at a density of 5×10⁶ cells/plate in 6 cm platesin DMEM containing 10% fetal calf serum 10 μg/ml penicillin-streptomycinand 2 mM glutamine one day prior to treatment. The media was thenreplaced with DMEM containing increasing concentrations of Dex eitheralone (◯) or together with 2 ng/ml LF in the presence of 500 ng/ml PA(●) or with 10 ng/ml LF in the presence of 500 ng/m PA (▴). After 18hours the cells were lysed by sonication and TAT activity assayed. Themean and standard deviations are shown and a one-way ANOVA followed by aBonferroni post hoc test was performed.

FIGS. 5A, 5B, 5C, and 5D are four graphs showing a comparison of theeffects of LeTx and inhibitors of MEK1 and JNK pathways on the responseof a Dex-induced GRE luciferase and a constitutive luciferase. Cos7cells were transfected with SVGR and (GRE)₂-TK luc (●) or with SVGR andthe constitutive luciferase vector, pGL3 control (Promega) (□) andtreated with 100 nM dexamethasone, and increasing concentrations LF with500 ng/ml PA (FIG. 5), or increasing concentrations of the MEK1inhibitors, PD98059 (FIG. 5B), and U0126 (FIG. 5C) or the JNK inhibitor,SP600126 (FIG. 5D). Means and standard deviations are shown and data wasanalyzed using a two-way ANOVA followed by a Scheffe post hoc test.

FIGS. 6A, 6B, 6C, 6D, 6E, and 6F are a set of graphs (FIGS. 6A, 6C and6E) and digital images of gels (FIGS. 6B, 6D, and 6F) showing the effectof p38 MAP kinase inhibitors on the response of a Dex-induced GREluciferase and a constitutive luciferase and on inhibition of p38. Cos7cells were transfected with SVGR and (GRE)₂-TK luc (▪) or with SVGR andthe constitutive luciferase vector, pGL3 control (□) and treated 100 nMdexamethasone, and increasing concentrations of the p38 MAP kinaseinhibitors, SB203580 (FIG. 6A), SB220025 (FIG. 6C) and p38 MAP kinaseinhibitor (FIG. 6E). Means and standard deviations are shown and datawas analyzed using a two-way ANOVA followed by a Scheffe post hoc test.Cos7 cells were pre-treated for 30 nin with various concentrations ofSB203580 (FIG. 6B), SB220025 (FIG. 6D) or p38 MAP kinase inhibitor (FIG.6F) and then further incubated with 10 μg/ml anisomycin for 30 min.Proteins were then subjected to SDS-PAGE and Western blotting using ananti-phospho-p38 antibody.

FIGS. 7A, 7B, 7C, and 7D are a set of four graphs showing the effects ofthe LeTx on hormone-induced activity of other nuclear hormone receptorsin cos7 cells. Cos7 cells were transfected as described for FIG. 1.except that 20 ng expression plasmids for MR (FIG. 7A), ERα (FIG. 7B),ERβ (FIG. 7C) or PR-B (FIG. 7D) were used. One hundred ng of the fireflyluciferase reporters, GRE-luc (FIG. 7A), 100 ng ERE-luc (FIGS. 7B and7C) or pHr-luc (FIG. 7D) were used. After transfection, the media wasreplaced with DMEM containing 10% charcoal-stripped media and 100 nMaldosterone (FIG. 7A), 1 nM 17β-estradiol (FIG. 7B), 100 nM17β-estradiol (FIG. 7C), or 100 nM progesterone (FIG. 7D), eithercontaining increasing concentrations of LF alone (▪) or in the presenceof 500 ng/ml PA (□). After 24 hours the cells were lysed and dataanalyzed as described earlier. The mean and standard deviation of fiveexperiments are shown.

FIG. 8 is a digital image of a gel showing that LF and PA do not affectGR binding to a GRE probe in a gel shift experiment Twenty-five μg ofGR-transfected cos7 cytosol was incubated with a [³²P] labeled GRE probein the presence of 40 fold excess unlabeled probe as a competitor orwith 5, 10 or 50 ng/ml LF, 10, 50 or 500 ng/ml PA, or with 5, 10 or 50ng/ml LF in the presence of 500 ng/ml PA. The samples were run on a 40%Tris-borate-EDTA (TBE) acrylamide gel and visualized by autoradiography.

FIG. 9 is a graph showing, that PA and/or LF do not prevent [³H]dexamethasone binding to GR transfected cos7 cell cytosol preparations.One hundred μg GR transfected cos7 cytosol was incubated overnight with10 nM [³H] dexamethasone in the presence or absence of 500 fold excessunlabeled dexamethasone and in the presence of 1 μM RU486, 500 ng/ml PA,50 ng/ml LF or 500 ng/ml PA+50 ng/ml LF. Bound was separated from freeand specific binding calculated. The percent specific binding incomparison to dexamethasone alone is shown.

FIG. 10 is a graph showing that RU486 can fully repressdexamethasone-induced GR transactivation and progesterone-induced PR-Btransactivation in cos7 cells even in the presence of LeTx. Cos7 cellswere transfected with SVGR and (GRE)₂-TK luc or PR-B and pLTR luc andthen treated with 100 nM dexamethasone or progesterone in the presenceof 2 ng/ml LF+500 ng/ml PA and increasing concentrations of RU486(maximum 1 μM). Relative luciferase values were measured.

FIG. 11 is a graph showing LeTx repression of dexamethasone inducedtyrosine aminotransferase (TAT) in mouse livers. BALB/cJ mice wereinjected with LeTx and 30 minutes later with Dex. After six and twelvehours liver TAT activity was assayed. Means and standard deviations ofsix to ten animals are shown and a two-way ANOVA followed by a Scheffepost hoc test was performed.

FIG. 12 is a schematic diagram showing the structure of the variousMR/GR chimeras and an indication as to whether these are repressed byLeTx on the (GRE)₂ TK luc promoter.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO:1 is the amino acid sequence of human immunodeficiency virus(HIV)-1 Tat protein.

DETAILED DESCRIPTION

I. Abbreviations

-   -   ANOVA: analysis of variance    -   AR: androgen receptor    -   ATP: adenosine triphosphate    -   DBD: DNA binding domain    -   Dex: dexamethasone    -   DMEM: Dulbecco's modified Eagle's medium    -   ER: estrogen receptor    -   EDTA: ethylenediaminetetraacetic acid    -   GR: glucocorticoid receptor    -   GRE: glucocorticoid response element    -   GRE-TK luc: glucocorticoid response element-luciferase reporter        construct    -   GS: glutamine synthase    -   HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   HTC: hepatoma cell line    -   IL-6: interleukin-6    -   IR: icosanoid receptor    -   LBD: ligand binding domain    -   LeTx: lethal toxin    -   LF: lethal factor    -   LPS: lipopolysaccharide    -   Luc: luciferase    -   MAPK: MAP Kinase    -   μg: microgram    -   μl: microliter    -   μM: micromolar    -   MR: mineralocorticoid receptor    -   MTT: 3,[4,5-dimethylthiazol-2-yl]-2,5-phenyltetrazolium bromide    -   NFκB: nuclear factor kappa B    -   ng: nanogram    -   nM: nanomolar    -   PA: protective antigen    -   PBS: phosphate buffered saline    -   PEPCK: phosphoenolpyruvate carboxykinase    -   PPAR: peroxisome receptor    -   PR-B: progesterone B receptor    -   PVDF: polyvinylidene fluoride    -   RAR: retinoid receptor    -   RU 486: mifepristone    -   RXR: retinoid receptor    -   SDS-PAGE: sodium dodecyl sulphate polyacrylamide-gel        electrophoresis    -   SVGR: SV glucocorticoid receptor    -   TAT: tyrosine aminotransferase    -   TBE: Tris-borate-EDTA    -   TR: thyroid hormone receptor    -   TNF-α: tumor necrosis factor-α    -   VDR: vitamin D receptor    -   XPAR: peroxisonie receptor        II. Description of Several Specific Embodiments

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Nuclear Hormone Receptors

Nuclear hormone receptors comprise a superfamily of proteins thatincludes receptors for glucocorticoids (GRs), androgens (ARs),mineralocorticoids (MRs), progestins (PRs), estrogens (ERs), thyroidhormones (TRs), vitamin D (VDRs), retinoids (RARs and RXRs), peroxisomes(XPARs and PPARs) and icosanoids (IRs). In addition, “orphan receptors,”such as steroid and thyroid receptors, are structurally related toclassic nuclear hormone receptors and are considered part of the nuclearhormone receptor superfamily. Unlike integral membrane receptors andmembrane-associated receptors, nuclear hormone receptors are located inthe cytoplasm or nucleus of eukaryotic cells.

Nuclear hormone receptors are specifically bound and activated byphysiologically important small molecule ligands. Ligands of nuclearhormone receptors include native hormones, such as progesterone,estrogen and testosterone, vitamins, as well as synthetic derivativecompounds, such as medroxyprogesterone acetate, diethylstilbesterol and19-nortestosterone. Nuclear hormone receptor ligands, when present in aphysiological compartment surrounding a cell, pass through the outercell membrane and bind to their cognate receptor with high affinity(commonly in the 0.01-20 nM range) to create an activatedligand/receptor complex. This complex translocates to the cell's nucleuswhere it binds to a specific gene or genes present in the cell's DNA.Once bound to DNA, the ligand/receptor complex modulates transcriptionof target genes and thereby regulates expression of specific proteinsencoded by the target genes.

The activated nuclear hormone receptor/ligand complex functions toinduce certain genes to initiate or increase transcriptional activity,and/or to suppress activity of other genes. Modulation of nuclearhormone receptor activity can therefore involve activation or inhibitionof receptor function, which in turn can involve increased or decreasedactivities of gene induction and/or suppression. A compound that mimicsthe effect of a native ligand of a nuclear hormone receptor is referredto as an “agonist,” while a compound that inhibits the effect of anative ligand is called an “antagonist.”

Ligands to the steroid receptors are known to play an important role inhealth of both women and men. Excesses or deficiencies of these ligandscan have profound physiological consequences. For example, an excess ofthe GR ligand glucocorticoid results in Cushing's Syndrome, whileglucocorticoid deficiency is associated with Addison's Disease. Thenative ligand progesterone in females, as well as synthetic analogues,such as norgestrel (18-homonorethisterone) and norethisterone(17α-ethinyl-19-nortestosterone), are effective in birth controlformulations, typically in combination with the female hormone estrogenor synthetic estrogen analogues, as modulators of both PR and ER. On theother hand, antagonists to PR are useful in treating hormone dependentcancers of the breast, ovaries, and uterus, and certain non-malignantconditions such as uterine fibroids and endometriosis, a leading causeof infertility in women. Similarly, AR antagonists, such as cyproteroneacetate and flutamide have proved useful in the treatment of hyperplasiaand cancer of the prostate.

The effectiveness of known modulators of steroid receptors is oftencompromised by their undesired side-effect profile, particularly afterlong-term administration. For example, the effectiveness of progesteroneand estrogen agonists, such as norgestrel and diethylstilbesterolrespectively, as female birth control agents must be weighed against theincreased risk of breast cancer and heart disease to women taking suchagents. Similarly, the progesterone antagonist, mifepristone (RU486), ifadministered for chronic indications, such as uterine fibroids,endometriosis and certain hormone-dependent cancers, could lead tohomeostatic imbalances in a patient due to its inherent cross-reactivityas a GR antagonist. Accordingly, identification of additional compoundsand methods for modulating activity of nuclear hormone receptors will beof significant value in the treatment of a wide range of diseases.

The glucocorticoid receptor (GR) is present in glucocorticoid responsivecells where it resides in the cytosol in an inactive state untilstimulated by a GR agonist. Upon stimulation the receptor translocatesto the cell nucleus where it specifically interacts with DNA and/orprotein(s) and regulates transcription in a glucocorticoid responsivemanner. Two examples of proteins that interact with the glucocorticoidreceptor are the transcription factors, API and NFκ-B. Such interactionsresult in inhibition of API- and NF κ-B-mediated transcription and arebelieved to be responsible for some of the anti-inflammatory activity ofendogenously administered glucocorticoids. In addition, glucocorficoidsmay also exert physiologic effects independent of nuclear transcription.

Biologically relevant glucocorticoid receptor agonists include cortisoland corticosterone. Many synthetic glucocorticoid receptor agonistsexist including dexamethasone, prednisone, prednisolone,methylprednisolone, and trimcinolone. Glucocorticoid receptorantagonists, for example RU486, typically bind to the receptor andprevent glucocorticoid receptor agonists from binding and elicitingGR-mediated events.

The search for effective nuclear hormone receptor agonists andantagonists and related methods for modulating nuclear hormone receptoractivity remains an important objective of academic, medical andindustry research. In this context, U.S. Pat. No. 5,767,113 disclosescertain synthetic steroid compounds that are reportedly useful forconcurrently activating glucocorticoid-induced response and reducingmultidrug resistance. Published European Patent Application 0 683 172,published Nov. 11, 1995, discloses certain11,21-bisphenyl-19-norpregnane derivatives reportedly havinganti-glucocorticoid activity useful to treat or preventglucocorticoid-dependent diseases. International Publication No. WO98/26783, published Jun. 25, 1998, discloses the use of certain steroidcompounds with anti-glucocorticoid activity for prevention or treatmentof psychoses or addictive behavior. International Publication No. WO98/27986, published Jul. 2, 1998, discloses methods for treatingnon-insulin dependent Diabetes Mellitus (NIDDM), or Type II Diabetes, byadministering a combination of treatment agents exhibitingglucocorticoid receptor type I agonist activity and glucocorticoidreceptor type II antagonist activity. Treatment agents such as certainsteroid compounds having both glucocorticoid receptor type I agonistactivity and glucocorticoid receptor type II antagonist activity arealso disclosed. International Publication No. WO 98/31702, publishedJul. 23, 1998, discloses certain 16-hydroxy-11-(substitutedphenyl)estra-4,9-diene derivatives reportedly useful in treatment orprophylaxis of glucocorticoid dependent diseases or symptoms. PublishedEuropean Patent Application 0 903 146, published Mar. 24, 1999, reportsthat the steroid 21-hydroxy-6,19-oxidoprogesterone (21OH-6OP) is aselective antiglucocorticoid useful for the treatment of diseasesassociated with an excess of glucocorticoids in the body, such as theCushing's syndrome or depression. Additional disclosures pertaining tothe identification and utility of nuclear hormone receptor agonists andantagonists are provided in U.S. Pat. No. 3,683,091; Japanese PatentApplication, Publication No. 45014056, published May 20, 1970; JapanesePatent Application, Publication No. 6-263688, published Sep. 20, 1994;International Publication No. WO 95/10266, published Apr. 20, 1995;Japanese Patent Application, Publication No. 45-36500, published Nov.20, 1970; European Patent Application, Publication No. 0 188 396,published Jul. 23, 1986; Japanese Patent 09052899, dated Feb. 25, 1997;and U.S. Pat. No. 5,696,127. All of the above cited patents andpublications are incorporated herein by reference.

Although there are compositions and methods proposed in the art formodulating nuclear hormone receptor activity and thereby amelioratingdisease mediated directly or indirectly by the action of nuclear hormonereceptors, there is a continuing need for and a continuing search in thefield for additional and more effective compositions and methods tosatisfy these objectives. Thus, the identification of compounds andmethods that effectively modulate nuclear hormone receptor activity withminimal side effects remains an important objective in the art. Asdisclosed herein, bacterial products can be used in methods to identifycompounds that modulate nuclear hormone receptor activity. In onespecific, non-limiting example, the bacterial product is from anthrax.

Anthrax

Anthrax is a zoonotic illness that has been recognized for a long periodof time. In the 1870s, Robert Koch demonstrated for the first time thebacterial origin of a specific disease, with his studies on experimentalanthrax, and also discovered the spore stage that allows persistence ofthe organism in the environment. Shortly afterward, Bacillus anthraciswas recognized as the cause of woolsorter disease (inhalationalanthrax). Bacillus anthracis is a large, gram-positive, sporulating rod,with square or concave ends.

Human cases of anthrax are invariably zoonotic in origin, with noconvincing data to suggest that human-to-human transmission has evertaken place. Primary disease takes one of three forms: (1) Cutaneous,the most common, results from contact with an infected animal or animalproducts; (2) Inhalational is much less common and a result of sporedeposition in the lungs, while (3) Gastrointestinal is due to ingestionof infected meat. Most literature cites cutaneous disease asconstituting the large majority (up to 95%) of anthrax cases.

Anthrax disease occurs when spores enter the body, germinate to thebacillary form, and multiply. In cutaneous disease, spores gain entrythrough cuts, abrasions, or in some cases through certain species ofbiting flies. Germination is thought to take place in macrophages, andtoxin release results in edema and tissue necrosis but little or nopurulence, probably because of inhibitory effects of the toxins onleukocytes. Generally, cutaneous disease remains localized, although ifuntreated it may become systemic in up to 20% of cases, withdissemination via the lymphatics. In the gastrointestinal form, B.anthracis is ingested in spore-contaminated meat, and may invadeanywhere in the gastrointestinal tract. Transport to mesenteric or otherregional lymph nodes and replication occur, resulting in dissemination,bacteremia, and a high mortality rate. As in other forms of anthrax,involved nodes show an impressive degree of hemorrhage and necrosis.

The pathogenesis of inhalational anthrax is more fully studied andunderstood. Inhaled spores are ingested by pulmonary macrophages andcarried to hilar and mediastinal lymph nodes, where they germinate andmultiply, elaborating toxins and overwhelming the clearance ability ofthe regional nodes. Bacteremia occurs, and death soon follows.Penicillin remains the drug of choice for treatment of susceptiblestrains of anthrax, with ciprofloxacin and doxycycline employed assuitable alternatives. Some data in experimental models of infectionsuggest that the addition of streptomycin to penicillin may also behelpful. Penicillin resistance remains extremely rare in naturallyoccurring strains, however the possibility of resistance should besuspected in a biological warfare attack. More severe forms of anthraxrequire intensive supportive care and have a high mortality rate despiteoptimal therapy. The use of anti-anthrax serum, while no longeravailable for human use except in the former Soviet Union, was thoughtto be of some use in the preantibiotic era, although no controlledstudies were performed.

Death from anthrax is reported to result from systemic shock resemblingLPS-induced toxic shock (P. Hanna, J. Appl. Microbiol. 87:285, 1999; P.C. Hanna et al., Trends Microbiol. 7:180, 1999), although the role ofinflammatory cytokines in this process has been questioned (J. L. Erwinet al., Infect. Immun., 69:1175, 2001).

A “bacterial product” is a compound produced by a bacteria, such as aprotein, superantigen, toxin or a polysaccharide. An exemplary bacterialproduct is a bacterial wall protein, soluble bacterial protein, orlipopolysaccharide The virulence of B. anthracis is dependent on twobacterial products, both of which are toxins, lethal factor (LF) andedema, as well as on the bacterial capsule. The importance of a toxin inanthrax pathogenesis was demonstrated in the early 1950s, when sterileplasma from anthrax-infected guinea pigs caused disease when injectedinto other animals (Smith et al., Nature, 173:869-870, 1954). It hassince been shown that the anthrax toxins are composed of three entities,which in concert lead to some of the clinical effects of anthrax(Stanley et al., J. Gen. Microbiol., 26:49-66, 1961; Beall et al., J.Bacteriol., 83:1274-1280, 1962). The first of these, protective antigen(PA), is an 83 kD protein so named because it is the main protectiveconstituent of anthrax vaccines. PA binds to the anthrax toxin receptor(ATR) on target cells and is then proteolytically cleaved by the enzymefurin of a 20 kd fragment (K. A. Bradley et al., Nature, 414:225, 2001;K. R. Klimpel et al., Proc. Natl. Acad. Sci. U.S.A., 89:10277, 1992).

The smaller cleaved 63 kD PA remnant (PA₆₃) oligomerizes features anewly exposed, second binding domain and binds to either EF, an 89 kDprotein, to form edema toxin, or LF, a 90 kD protein, to form lethaltoxin (LeTx) (Leppla et al., Salisbury Med. Bull. Suppl., 68:4143,1990), and the complex is internalized into the cell by (Y. Singh etal., Infect. Immun. 67:1853, 1999; A. M. Friedlander, J. Biol. Chem,261:7123, 1986). From these endosomes, the PA₆₃ channel enablestranslocation of LF and EF to the cytosol by a pH- and voltage-dependantmechanism (J. Zhao et al., J. Biol. Chem., 270:18626, 1995; J. Wesche etal., Biochemistry, 37:15737, 1998; R. O. Blaustein et al., Proc. Natl.Acad. Sci. U.S.A., 86:2209, 1989).

Edema factor, a calmodulin-dependent adenylate cyclase, acts byconverting adenosine triphosphate to cyclic adenosine monophosphate.Intracellular cyclic adenosine monophosphate levels are therebyincreased, leading to the edema characteristic of the disease (Leppla etal., Proc. Natl. Acad. Sci. USA, 79:3162-3166, 1982).

It is the lethal toxin produced by Bacillus anthracis that causes thedeath of infected hosts (C. Pezard et al., Infect. Immun., 59:3472,1991). Lethal toxin has been demonstrated to lyse macrophages at highconcentration, while inducing the release of tumor necrosis factor andinterleukin 1 at lower concentrations (Hanna et al., Proc. Natl. Acad.Sci. USA, 90:10198-10201, 1993; Freidlander J. Biol. Chem.,261:7123-7126, 1986). It has been shown that a combination of antibodiesto interleukin 1 and tumor necrosis factor was protective against alethal challenge of anthrax toxin in mice, as was the human interleukin1 receptor antagonist (Hanna et al., supra). Macrophage-depleted micewere shown to resist lethal toxin challenge, but to succumb whenmacrophages were reconstituted. The genes for both the toxin and thecapsule are carried by plasmids, designated pX01 and pX02, respectively.

Although anthrax vaccination dates to the early studies of Greenfieldand Pasteur, the “modern” era of anthrax vaccine development began witha toxin-producing, unencapsulated (attenuated) strain in the 1930s.Administered to livestock as a single dose with a yearly booster, thevaccine was highly immunogenic and well tolerated in most species,although somewhat virulent in certain species. This preparation isessentially the same as that administered to livestock around the worldtoday.

The first human anthrax vaccine was developed in the 1940s fromnonencapsulated strains. This live spore vaccine is administered byscarification with a yearly booster. Studies show a reduced risk of 5-to 15-fold in occupationally exposed workers (Shlyakhov et al., Vaccine,12:727-730, 1994). British and U.S. vaccines were developed in the 1950sand early 1960s, with the U.S. product an aluminum hydroxide-adsorbed,cell-free culture filtrate of an unencapsulated strain (V770-NP1-R), andthe British vaccine an alum-precipitated, cell-free filtrate of a Sternestrain culture. The U.S. vaccine has been shown to induce high levels ofantibody only to protective antigen, while the British vaccine induceslower levels of antibody to protective antigen but measurable antibodiesagainst lethal factor and edema factor (Turnbull et al., Infect.Immunol. 52:356-363, 1986; Turnbull et al., Med. Microbiol. Immunol.177:293-303, 1988). Neither vaccine has been examined in a humanclinical efficacy trial. A high number of the recipients of the vaccinehave reported some type of reaction to vaccination. Manufacturerlabeling for the current Michigan Department of Public Health anthraxvaccine adsorbed (AVA) product cites a 30% rate of mild local reactionsand a 4% rate of moderate local reactions with a second dose.

One significant limitation on the use of vaccines is that existingvaccines provide no protection against a number of strains of B.anthracis. Recent incidents, such as the suspected use of biological andchemical weapons during the Persian Gulf War, underscore the threat ofbiological warfare either on the battlefield or by terrorists. Anthraxhas been the focus of much attention as a potential biological warfareagent for at least six decades, and modeling studies have shown thepotential for use in an offensive capacity. Dispersal experiments withthe simulant Bacillus globigii in the New York subway system in the1960s suggested that release of a similar amount of B. anthracis duringrush hour would result in 10,000 deaths. On a larger scale, the WorldHealth Organization estimated that 50 kg of B. anthracis released upwindof a population center of 500,000 would result in up to 95,000fatalities, with an additional 125,000 persons incapacitated (Huxsoll etal., JAMA, 262:677-679, 1989). Both on the battlefield and in aterrorist strike, B. anthracis has the attribute of being potentiallyundetectable until large numbers of seriously ill individuals presentwith characteristic signs and symptoms of inhalational anthrax. Giventhese findings, efforts to prevent the disease or to ameliorate or treatits effects are of major importance.

Modulation of Activity of a Nuclear Hormone Receptor by a BacterialProduct

Compounds, pharmaceutical compositions, and methods for modulatingprocesses mediated by a nuclear hormone receptor are disclosed herein.In several embodiments, the nuclear hormone receptor is a glucocorticoidreceptor (GR), androgen receptor (AR), mineralocorticoid receptor (MR),progestin receptor (PR), estrogen receptor (ER), thyroid hormonereceptor (TR), vitamin D receptor (VDR), retinoid receptor (RAR or RXR),peroxisome receptor (XPAR or PPAR), or icosanoid receptor (IRs). Inother specific embodiments the receptor is an orphan receptor, forexample a steroid receptor and/or thyroid receptor.

It is disclosed herein that a bacterial product, specifically abacterial toxin affects the activity of the glucocorticoid receptor(GR). In one example, the toxin is the anthrax lethal factor (LF)produced by Bacillus anthracis. In one example, this activity of theexemplary bacterial toxin LF can be assayed using a reporter system. Aspecific non-limiting example of a reporter system is a transientgluccocorticoid responsive element (GRE)-luciferase transfection system,which establishes GR repression by LF to a level of 50%, and at very lowconcentrations as low as 1.5-2.0 ng/ml. In cellular systems when LF isexogenously applied this effect occurs only in the presence of theanthrax protective antigen (PA), a protein produced by the anthraxbacteria that is essential for transport of LF into cells. However, incertain embodiments, LF alone may mediate GR repression and relatedeffects when delivered internally in cells (such as when cells have beentransduced with a polynucleotide encoding LF to express the proteinendogenously).

Bacterial products of use include bacterial wall proteins and otherproducts (such as streptococcal or staphylococcal cell walls andlipopolysaccharide (LPS), and soluble antigens of bacteria. The productsof interest can exert various effects on infected hosts, for example bycausing damage to cell membranes, inhibition of protein synthesis,activation of second messenger pathways, activation of immune responses,and/or degradation of host proteins (such as by functioning as ametalloprotease). In specific embodiments, the bacterial product is abacterial toxin. As used herein bacterial toxins include bacterialproducts that mediate toxic effects, inflammatory responses, stress,shock, chronic sequelae, or mortality in a susceptible host. Exemplarybacterial toxins are anthrax LF and LeTx, and metalloenzymes ofClostridium tetanus and C. botulinum bacteria. In one embodiment, thebacterial product is not endotoxin. In other embodiments, the bacterialproduct is a bacterial antigen, for example a pyrogenic toxinsuperantigen (PTSAg) (such as a staphylococcal enterotoxin, exfoliativetoxin, or toxic-shock toxin. In other specific embodiment, the bacterialproduct is a toxin, but is not endotoxin. Table 1, below, sets forth anexemplary list of candidate bacterial products characterized genericallyas bacterial toxins that will find use within the methods andcompositions disclosed herein (see also review by Schmidtt et al.,Emerg. Infect. Dis., 5:224-234, 1999).

LF and other bacterial products can be selected for use by their abilityto interact with one or more nuclear hormone receptors, directly orindirectly (such as by interacting with a co-factor of a nuclear hormonereceptor), in a manner that modulates activity of the receptor(s).Often, receptor modulation in this context will suppress or amplify aninflammatory response, autoimmune symptom, or other adverse symptoms inthe subject, for example by repressing the anti-inflammatory effects ofthe glucocorticoids. In the case of anthrax, the inflammatory, toxicand/or lethal effects of Bacillus anthracis may be caused at least inpart by antagonism/repression of the glucocorticoid receptor bybacterial products. In one emobidment, this interaction is not throughthe ligand binding domain.

As discussed briefly above, anthrax toxins are composed of threeproteins: lethal factor (LF), protective antigen (PA) and edema factor(EF) (S. H. Leppla, Comprehensive Sourcebook of Bacterial ProteinToxins. ed., 243-63, 1999; S. H. Leppla Bacterial Protein Toxins. ed.,445-72, 2000). PA facilitates entry of LF and EF into cells. LF is a 90kD metalloprotease, for which the crystal structure has recently beendetermined (A. D. Pannifer et al., Nature, 414:229-33, 2001). All threegenes are encoded by the plasmid pXO1 (M. Mock et al., Annu. Rev.Microbiol., 55:647-71, 2001). Together, LF and PA constitute the lethaltoxin (LeTx), and EF and PA the edema toxin.

Studies directed at the mechanism of action of LeTx have mainly focusedon its action in cleaving MAPKK. While this action effectively andrapidly removes this important signal transduction molecule, evidence ofsome transient activation of the system, such as phosphorylation of ERK,has also been observed (R. Pellizzari et al.,. Int. J. Med. Microbiol.,290:421-7, 2000). Nonetheless, LF resistant and susceptible cell linesshow equal MAPKK proteolysis by LeTx (R. Pellizzari et al., Int. J. Med.Microbiol., 290:421-7, 2000; P Pellizzari et al., FEBS letters.462:199-204, 1999). Thus, while LeTx does cleave MAPKK, other oradditional biological activities could be needed to cause its toxic andlethal effects. Such an activity, namely the interaction with a nuclearreceptor, is demonstrated herein.

Synopses

TABLE 1 Characteristics of bacterial toxins^(a) Toxin implicatedOrganism/Toxin Mode of Action Target Disease in disease

Damage membranes Aeromonas hydrophila/aerolysin Pore-former GlycophorinDiarrhea (yes) Clostridium perfringens/ Pore-former Cholesterol Gasgangrene^(c) ? perfringolysin O Escherichia coli/hemolysin^(d)Pore-former Plasma membrane UTIs (yes) Listeria monocytogenes/Pore-former Cholesterol Foodborne (yes) listeriolysin O systemicillness, meningitis Staphyloccocus aureus/a-toxin Pore-former Plasmamembrane Abcesses^(c) (yes) Streptococcus Pore-former CholesterolPneumonia^(c) (yes) pneumoniael/pneumolysin Streptococcus Pore-formerCholesterol Strep throat, Sf^(c) ? pyogenesl/streptolysin O Inhibitprotein synthesis Corynebacterium ADP-ribosyltransferase Elongationfactor 2 Diphtheria yes diphtheriae/diphtheria toxin E. coli/Shigelladysenteriae/ N-glycosidase 285 rRNA HC and HUS yes Shiga toxinsPseudomonas aeruginosa/ ADP-ribosyltransferase Elongation factor 2Pneumonia^(c) (yes) exotoxin A Activate second messenger pathways E.coli CNF Deamidase Rho G-proteins UTIs ? LT ADP-ribosyltransferaseG-proteins Diarrhea yes ST^(d) Stimulates guanylate guanylate cyclaseDiarrhea yes cyclase receptor CLDT^(d) G2 block Unknown Diarrhea (yes)EAST ST-like? Unknown Diarrhea ? Bacillus anthracis/edema factorAdenylate cyclase ATP Anthrax yes Bordetella pertussis/ Deamidase RhoG-proteins Rhinitis (yes) dermonecrotic toxin pertussis toxinADP-ribosyltransferase G-protein(s) Pertussis yes Clostridiumbotulinum/C2 toxin ADP-ribosyltransferase Monomeric G-actin Botulism ?C. botulinum/C3 toxin ADP-ribosyltransferase Rho G-protein Botulism ?Clostridium difficilel toxin A Glucosyltransferase Rho G-protein(s)Diarrhea/PC (yes) toxin B Glucosyltransferase Rho G-protein(s)Diarrhea/PC ? Vibrio cholerae/cholera toxin ADP-ribosyltransferaseG-protein(s) Cholera yes Activate immune response S. aureus/Superantigen TCR and MHC B Food poisoning^(c) yes enterotoxinsexfoliative toxins Superantigen (and serine TCR and MHC II SSS^(c) yesprotease?) toxic-shock toxin Superantigen TCR and MHC II TSS^(c) yes S.pyogerces/pyrogenic Superantigens TCR and MHC II SF/TSS^(c) yesexotoxins Protease B. anthracis/lethal factor MetalloproteaseMAPKK1/MAPKK2 Anthrax yes C. botulinum/neurotoxinsA-GZinc-metalloprotease VAMP/ Botulism yes synaptobrevin, SNAP-25, syntaxinClostridium tetani/tetanus toxin Zinc-metalloprotease VAMP/synaptobrevinTetanus yes^(a)Abbreviations: CNF, cytotoxic necrotizing factor; LT, heat-labiletoxin; ST, heat-stable toxin; CLDT, cytolethal distending toxin; EAST,enteroaggregative E. coli heat-stable toxin; TCR, T-cell receptor; MHCII, major histocompatibility complex class II; MAPKK, mitogen-activatedprotein kinase kinase; VAMP, vesicle-associated membrane protein;SNAP-25, synaptosomal associated protein; UTI, urinary tract infection;HC, hemorrhagic colitis; HUS, hemolytic uremic syndrome; PC,# antibiotic associated pseudomembranous colitis; SSS, scalded skinsyndrome; SF, scarlet fever; TSS, toxic-shock syndrome.^(b)Yes strong causal relationship between toxin and disease; (yes),role in pathogenesis has been shown in animal model or appropriate cellculture; ?, unknown.^(c)Other diseases are also associated with the organism.^(d)Toxin is also produced by other genera of bacteria.

Radioligand competition studies detailed below indicate that neither LFnor PA, alone and/or together, competes with dexamethasone for bindingto the ligand binding site of GR, nor do they interfere with GR-GRE DNAbinding in electrophoretic mobility shift assays (EMSAs). Thus, anthraxlethal toxin (LeTx) and lethal factor (LF) specifically repressesactivation of glucocorticoid receptor in a dose-related,non-competitive, non-ligand or DNA-binding manner. This bacterialproduct can exert its effect on nuclear hormone receptor repressionthrough a cofactor involved in the interaction between nuclear hormonereceptors and the basal transcription machinery, and/or by acting itselfas a co-repressor. In one embodiment, the repression of GR is mediatedthrough the DNA binding domain (DBD), co-factor binding, or downstreampathways that interact with these domains of the receptor and the basaltranscription machinery.

In another embodiment, repression of a nuclear hormone receptor by abacterial product is not mediated by inhibition of a MEK1 or MAPKKpathway. This finding contrasts with many reports suggesting that thetoxic or shock-related activities of LF are mediated by LF's proposedmetalloprotease function and a putative degradation by LF of proteinsinvolved in the MEK1 and/or MAPKK pathway(s). The MAPK pathway consistsof three separate pathways, MEK, SEK and p38 (Pellozzari et al., FEBSLett., 462:199, 1999; Pellozzari et al., J. Med. Microbiol. 290:421,2000).

The MEK and p38 pathways are known to be targets of LeTx. In theexamples below it is demonstrated that PD98059, an inhibitor of the MEKpathway, does not have any GR specific effect in a transienttransfection system. SB203580, an inhibitor of the p38 pathway also hasno effect on GR-mediated transactivation in a GRE-luciferase system.LeTx is known to degrade some proteins of the MAPK pathway includingMEK. The data presented herein indicate that MEK degradation alone doesnot determine LeTx sensitivity and that other factor(s) must beinvolved. As disclosed herein, repression of GR and other nuclearhormone receptor hormones is a factor in determining LeTx sensitivity.

In yet another embodiment, repression of a nuclear hormone receptor by abacterial product is not mediated by a change in the number of nuclearhormone receptors on a cell. Thus, cells treated with the bacterialproduct have substantially the same number of nuclear hormone receptorsas cells not treated with the bacterial product. In several example, thenumber of receptors does not change by more than about 1%, more thanabout 5%, more than about 10% or more than 25% upon treatment with thebacterial product (as compared to cells not treated with the bacterialproduct). In other examples, no statistically significant difference inthe number of receptors is observed following treatment with thebacterial product. One of skill in the art can readily identifyappropriate statistical analyses for this determination. Exemplarymethods for determining the number of nuclear hormone receptors on cellsare provided in the Examples section below.

LF activity is specific for some nuclear hormone receptors, whereasother bacterial products as disclosed herein will be specific for thesame or different receptors, or more generalized by acting to repress awider group of nuclear hormone receptors. In the case of anthrax LF andLeTx, this bacterial toxin is demonstrated herein to repress GR (Type 1GR) but not the mineralocorticoid (MR, Type II GR) receptor, and torepress estrogen receptor-α (Er-α) but not ER-β, and the progesteronereceptor B (PR-B).

Nuclear hormone receptor agonists and antagonists are useful toinfluence basic, life sustaining systems of the body, includingcarbohydrate, protein and lipid metabolism, electrolyte and waterbalance, and the functions of the cardiovascular, kidney, centralnervous, immune, skeletal muscle and other organ and tissue systems. Inthis regard, GR and MR modulators have proved useful in the treatment ofinflammation, tissue rejection, auto-immunity, hypertension, variousmalignancies, such as luekemias, lymphomas, and thyroid, breast andprostate cancers, Cushing's syndrome, glaucoma, obesity, rheumatoidarthritis, acute adrenal insufficiency, congenital adrenal hyperplasia,osteoarthritis, rheumatic fever, polymyositis, polyarteritis nodosa,granulomatous polyarteritis, allergic diseases such as urticaria, drugreactions and hay fever, asthma, a variety of skin diseases,inflammatory bowel disease, hepatitis and cirrhosis. Accordingly, inexemplary embodiments, GR and MR modulatory compounds are useful asimmuno stimulants and repressors, wound healing and/or tissue repairagents, catabolic/antianabolic activators, and as antibacterial oranti-viral agents (such as for treatment or prevention of symptomsrelated to anthrax, herpes simplex viral infection and relatedsymptoms). Additional diseases that may prove amenable to diagnosisand/or management using the methods and compositions disclosed hereininclude, but are not limited to, Parkinson's disease, cardiovasculardisease including restenosis, anxiety, depression, psychosis, variousviral infections, including HIV and HSV, proliferative andhyperproliferative disorders, including restenosis and psoriasis.

Autoimmune diseases or disorders that can be treated, prevented, and/ordiagnosed by polynucleotides, polypeptides, antibodies, and/or agonistsor antagonists of nuclear receptors include but are not limited to, oneor more of the following: systemic lupus erythematosus, rheumatoidarthritis, ankylosing spondylitis, multiple sclerosis, autoimmunethyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia,hemolytic anemia, thrombocytopenia, autoimmnune thrombocytopeni purpura,autoimmune neonatal thrombocytopenia, idiopathic thrombocytopeniapurpura, purpura a (such as Henloch-Scoenlein purpura),autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris,myasthenia gravis, Grave's disease (hyperthyroidism), andinsulin-resistant diabetes mellitus.

Additional disorders that are likely to have an autoimmune componentthat can be treated, prevented, and/or diagnosed using the methods andcompositions disclosed herein include, but are no limited to, type IIcollagen-induced arthritis, antiphospholipid syndrome, dermatitis,allergic encephalomyelitis, myocarditis, relapsing polychondritis,rheumatic heart disease, Neuritis, Uveitis Ophthalmia,Polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, AutoimmunePulmonary Inflammation, Autism, Guillain-Barre Syndrome, insulindependent diabetes mellitis, and autoimmune inflammatory eye.

Yet additional disorders that are likely to have an autoimmune componentthat can be treated, prevented, and/or diagnosed with the compositionsdisclosed herein include, but are not limited to, scleroderma withanti-collagen antibodies (often characterized, such as by nucleolar andother nuclear antibodies), mixed connective tissue disease (oftencharacterized, such as by antibodies to extractable nuclear antigens(for example, ribonucleoprotein)), polymyositis (often characterized,for example, by nonhistone ANA), pernicious anemia (often characterized,for example, by antiparietal cell, microsomes, and intrinsic factorantibodies), idiopathic Addison's disease (often characterized, forexample, by humoral and cell-mediated adrenal cytotoxicity, infertility(often characterized, for example, by antispermatozoal antibodies),glomerulonephritis (often characterized, for example, by glomerularbasement membrane antibodies or immune complexes), bullous pemphigoid(often characterized, for example, by IgG and complement in basementmembrane), Sjogren's syndrome (often characterized, for example, bymultiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)),diabetes mellitus (often characterized, for example, by cell-mediatedand humoral islet cell antibodies), and adrenergic drug resistance(including adrenergic drug resistance with asthma or cystic fibrosis)(often characterized, for example, by beta-adrenergic receptorantibodies).

Additional disorders that may have an autoimmune component that can betreated, prevented, and/or diagnosed with the compositions disclosedherein include, but are not limited to, chronic active hepatitis (oftencharacterized, for example by smooth muscle antibodies), primary biliarycirrhosis (often characterized, for example, by mitchondrialantibodies), other endocrine gland failure (often characterized, forexample, by specific tissue antibodies in some cases), vitiligo (oftencharacterized, for example, by melanocyte antibodies), vasculitis (oftencharacterized, for example, by Ig and complement in vessel walls and/orlow serum complement), post-MI (often characterized, for example, bymyocardial antibodies), cardiotomy syndrome (often characterized, forexample, by myocardial antibodies), urticaria (often characterized, forexample, by IgG and IgM antibodies to IgE), atopic dermatitis (oftencharacterized, for example, by IgG and IgM antibodies to IgE), asthma(often characterized, for example, by IgG and IgM antibodies to IgE),and many other inflammatory, granulamatous, degenerative, and atrophicdisorders.

For treatment and prevention of bacterial disease and associatedinflammatory, autoimmune, toxic (including shock), and chronic and/orlethal sequelae associated with bacterial infection a wide variety ofeffective compositions and methods are provided. In one embodiment, oneor more symptoms or associated effects of exposure to and/or infectionwith anthrax is/are prevented or treated by administration to amammalian subject at risk of acquiring or presenting with the symptom(s)of an effective amount of an agent that affects nuclear hormone receptoractivity. In exemplary embodiments, these treatment and prophylacticmethods and compositions employ drugs and other agents identifiedaccording to the methods herein to bypass or diminish blockade of anuclear hormone receptor mediated by a bacterial product (for example,LF blockade of the glucocorticoid receptor (GR), PR or other nuclearhormone receptor(s)). Alternative approaches to bypassing or reducingnuclear hormone receptor activation and/or repression involve, forexample in the case of anthrax, treatment with glucocorticoid or othernuclear hormone receptor agonists or antagonists, or with agents thatinteract with GR and LF/PA, or with agents that enhance or repress GRand/or other nuclear hormone receptor co-factors.

Therapeutic compositions and methods for prevention or treatment oftoxic or lethal effects of bacterial infection are applicable to a widespectrum of infectious agents. Non-lethal toxicities that will beameliorated by these methods and compositions include fatigue syndromes,inflammatory/autoimmune syndromes, hypoadrenal syndromes, weakness,cognitive symptoms and memory loss, mood symptoms, neurological and painsyndromes and endocrine symptoms.

These compositions and methods are also applicable to treatment andprevention of toxic effects of exposure to anthrax and/or relatedbacterial vaccines. Reports indicate that Gulf War syndrome symptoms offatigue, depression, inflammatory/autoimmune, weakness, memory loss,neurological, pain, endocrine and other symptoms, may be related tovaccination with anthrax vaccine. The currently available anthraxvaccine, derived from a bacterial cell filtrate of Bacillus anthraciscontains variable amounts of LF, and acute and chronic effects areprobably related to interactions of vaccine components with GR and/orits cofactors and other nuclear hormone receptors and/or theirco-factors. Thus, as disclosed herein, antagonists of GR can be used forthe prevention and treatment of side effects related to the anthraxvaccine, as well as a means to produce vaccine without or with lowerrisk of such side effects.

Additional embodiments are directed to diagnostic compositions andmethods to identify individuals at risk for toxic effects or long-termdeleterious effects of exposure to pathogenic bacteria, for exampleanthrax bacteria, and their cognate vaccines. Certain strains of rodentsshow enhanced susceptibility to lethal effects of exposure to anthrax.The disclosure herein implicates differences in characteristics, numberor regulation of GR, GR co-factors or other nuclear hormone receptorsand their co-factors for these strain differences. Identification andcharacterization of GR, its co-factors and other nuclear hormonereceptor co-factors according to the present disclosure will provideeffective tools for identifying individuals who may be genetically orotherwise predisposed to development of toxic lethal or long termchronic effects from exposure to bacterial pathogens and vaccinesdirected to them. Thus, in one embodiment a bacterial produced can beused to identify a subject having or at risk of developeing a disorder,such as a disorder associated with a cofactor of a nuclear hormonereceptor.

In additional aspects, the methods and compositions disclosed herein areuseful for identification of environmental agents, including otherbacterial products (for example, products of food-borne pathogens) thatmediate idiopathic inflammatory, autoimmune, fatigue, memory loss,endocrine and other syndromes. Certain individuals exposed to smallamounts of bacterial products, such as those derived from anthrax,presenting certain genetic or physiological backgrounds, are predisposedto development of chronic syndromes, including fatigue syndromes,inflammatory/autoimmune syndromes, hypoadrenal syndromes, weakness,cognitive symptoms and memory loss, mood symptoms, neurological and painsyndromes and endocrine symptoms. In this context, the methods andcompositions disclosed herein employed to detect, and alternatively totreat and/or prevent, such ubiquitous environmental exposures andassociated symptoms. For example, methods for screening for LF/PA-likebacterial products or other environmental agents that interact withnuclear hormone receptors or their co-factors in a manner associatedwith disease or other adverse symptoms or conditions in mammaliansubjects.

In one embodiment, LF and other bacterial products that specificallyblock, degrade or otherwise interact with one of the GR or other nuclearhormone receptor co-factors are employed as reagents in variousscreening methods to identify, for example whether a specific co-factoris involved in an important hormonal action. Certain screening methodswill “knock out” a subject cofactor (for example, in an engineered cellor knock-out mouse) in order to clarify the role of the co-factor inmediating receptor modulation and/or disease. As such, a large panel ofknown cofactors of nuclear hormone receptors will find utility and aretherefore incorporated within various embodiments of the compositionsand/or methods thereof. Exemplary members of this panel are set forthbelow in Table 2. TABLE 2 CO-ACTIVATORS OF NUCLEAR HORMONE RECEPTORSName Other names p160 family ERAP160 RIP160 SRC-1 N-CoA1 TIF2 GRIP1,SRC-2 p/CIP ACTR, RAC3, AIB-1, TRAM-1, SRC-3 p140 family p140 ERAP140,RIP140 p300 family p300 CBP P/CAF (ADA/SAGA) complex PCAF GCN5Tra1/TRRAP PAF65α PAF65β TAF31 hTAF_(II)31 TAF30 hTAF_(II)30 TAF20hTAF_(II)20 hAda2 hSPT3 hAda3 hTAF_(II)15 Basal Transcriptionalmachinery TFIID TFIIH TFIIE TEIIF TBP TBP-associated factors (TAFs)TAF250 TAF130 TAF28 TAF18 TAF55 TAF150 TAF70 TAF31 TAF20 TAF100 TAF30Universal stimulatory activity (USA) NC2 - subunits Dr1 and Drap1 PC2PC4 Mediator/SRB Med10 Med7 Med6 Med1 Med2 Med3 Med4 Med5 Gal11 Sin4Rgr1 Rox3 NAT complex SRB10/CDK8 Srb7 Srb10 Rgr1 Med6 P230 P150 P140HSur2 P95 P90 P70 P56 Cdk8 P45 P37 P36p33 P31 Cyclin C P30 P23 P22 P21P17 P14 SMCC complex SRB11/cyclinC CRSP complex Med7 Rgr1 CRSP200CRSP150 CRSP130 CRSP77 CRSP70 CRSP34 CRSP33 Drip complex DRIP205 DRIP240DRIP250 DRIP70 DRIP77 DRIP92 DRIP100 DRIP130 DRIP150 DRIP97 DRIP70-2Cdk8 DRIP36 DRIP34 DRIP33 hSrb7 hMed10 TRAP complex TRAP80 TRAP93 TRAP95TRAP97 TRAP100 TRAP150 TRAP170 TRAP220 TRAP230 TRAP240 hSrb10 hMed7hMed6 hTRF hSrb11 hSoh1 hSrb7 hNut2 ARC complex ARC250 ARC240 ARC205ARC150 ARC130 ARC105 TIG-1 ARC100 ARC92 ARC77 ARC70 ARC42 ARC36 ARC34ARC33 ARC32 NUA3 COMPLEX (YEAST) NuA4 complex (Yeast) E3Ubiquitin-protein ligases RPF-1 E6-AP ARNIP Histone MethyltransferasesCARM-1 PRMT-1 Suv39H1 G9a Set 9 Set 7 Chromatin modifying ATPasesSWI/SNF COMPLEX Brahma (BRM) Brahma-related gene-1 (BRG-1) hSNF2□ BRG-1associated factors (BAFs) INI1 BAF47 BAF155 BAF170 BAF57 BAF250 hSNF5INI1, BAF47 BAF60 BAF53 NURF complex NURF301 NURF140 NURF55 NURF38NURF215 NURD complex Mi-2β CHD4 Mi-2α CHD3 HDAC1 NURD63 HDAC2 NURD59RbAp48 NURD56 RbAp46 NURD55 MTA1/2 NURD70 MBD3 ACF complex Acf1 ISWICHRAC complex ACF1 hSNF2H hSNF2L RSF complex hSNF2h p325 TIF1 NSD-1Co-repressors RIP13 NCoR SMRT Sin3-A Rpd1 Sin3-B HDAC-1 Rpd3 HDAC-2HDAC-4 HDAC-5 HDAC-6 HDAC-7 HDAC-3 RbAp46/48 Mi-2 CHD4 MBD2 MeCP1 OthersSug1 Trip1 GRIP95 GRIP120 GRIP170 ARA₇₀ RIP80 CREBSee also, Glass et al., Curr. Opin. Cell Biol 9: 222-232, 1997; McKennaet al., Eudocrinology, 143: 2461-2465, 2002.

In other embodiments, LF or other bacterial products that specificallyblock, degrade or otherwise interact with one of the GR or other nuclearhormone receptor co-factors, antagonists or agonists are employed toinduce, amplify or increase or decrease expression of a particularco-factor with which the subject bacterial product interacts, forexample in a method or composition to treat or prevent toxicity mediatedby LF or another bacterial toxin. Within more detailed aspects, analogsor variants of LF and other bacterial products, and mimetics and drugsthat mimic one or more activities (for example, co-factor binding,co-factor degradation, hormone repression) of LF or another bacterialproduct, may be generated (such as by genetic engineering or chemicalmodification) to render the product non-toxic while retaining some orall of its function in altering nuclear hormone receptor activity (suchas to treat or prevent disease associated with elevated expression oractivation of a nuclear hormone receptor). Alternatively, analogs andvariants of bacterial products, as well as mimetics and drugs may bedeveloped by routine methods and identified using screening methodspresented herein, that block the binding or activity of a correspondingwild type bacterial product to thereby function, directly or indirectly,as an effective nuclear hormone receptor agonist. Such variants anddrugs based on LF or other bacterial toxins will often specificallyblock, degrade, stimulate or otherwise interact with one of the GR orother nuclear hormone receptor co-factors (co-activators orco-repressors), and thereby reduce or enhance the activity of thenuclear hormone receptor. These effects may mediate modulation ofactivity of one, or a plurality of, nuclear hormone receptors with whichthese co-factors interact. Thus, novel tools and methods are providedthat utilize a limited assemblage of ligand binding agents for blockingor enhancing activity of nuclear hormone receptors. The compositions andmethods disclosed herein further provide means to specifically andpartially reduce some but not all actions of nuclear hormone receptorhormones, for example when certain target co-factors are specificallyexpressed in certain tissues but not in others.

In yet additional embodiments, a recombinantly or chemically modifiedanalog, fragment or derivative of LF, or of another bacterial productdescribed herein, is employed in a vaccine or therapeutic formulation ormethod. Often, the modified analog, fragment or derivative will exhibitsubstantially reduced or enhanced activity as a modulator (such as arepressor or activator) of nuclear hormone receptor activity compared toa native or wild-type counterpart bacterial product. For example, amodified anthrax LF analog or fragment will exhibit a reduction orincrease in a level of GR repression or PR repression in an in vitro orin vivo assay of approximately 20%, 30%, 50%, 75% and up to 95% orgreater compared a control level of repression mediated by a native LFprotein (alone or complexed with PA). Other analogs and variants of LFor other selected bacterial products will alternatively or additionallyspecifically inhibit or block, or enhance, interactions of thecorresponding native bacterial product with a nuclear hormone receptor.For example, various analogs or variants of LF may competitively inhibitnative LF activity (such as cofactor binding, cofactor degradation,and/or GR or PR repression activity) or act as an LF agonist or mimeticin an in vitro or in vivo assay.

The various analogs, variants, derivatives and mimetics of bacterialproducts provided herein are useful for, inter alia, treatment and/orprevention of diseases, symptoms and conditions relating to bacterialinfection, inflammatory responses, and/or autoimmune disorders. In otherembodiments, analogs, variants, derivatives and mimetics of bacterialproducts are useful to provide more effective vaccine compositions andmethods, particularly to minimize adverse side effects that attendvaccination using a native or wild-type bacterial product. In oneexemplary embodiment, a mutant variant, truncated fragment, orchemically modified derivative of a LF protein is employed as atherapeutic or vaccine agent. The LF variant, fragment or derivativewill have substantially reduced or increased activity for nuclearhormone receptor modulation (for example, GR and/or PR repression). Atthe same time, the LF variant, fragment or derivative will exhibitsubstantial activity as an immunogen, and/or will inhibit, block orenhance (directly or indirectly) nuclear hormone receptor modulation bynative LF or LeTx.

Analogs, variants, derivatives and mimetics of bacterial products willtypically be effective to elicit an immune response in a mammaliansubject against a corresponding, native bacterial product, whereby thesubject will generate a humoral or cell-mediated immune response againstthe native product that is effective to prevent or reduce infection oralleviate one or more symptoms associated with infection by a pathogenexpressing the native product. For example, analogs, variants,derivatives and mimetics of LF and other bacterial products may begenerated (for example, by genetic engineering or chemical modification)to render the LF non-toxic. In certain embodiments, the bacterialproduct will be produced that exhibit increased or reduced activity ofthe analog, variant, derivative or mimetic for modulation of one or morenuclear hormone receptors (such as to have substantially reduced GR orPR repression activity). Typically, the analogs, variants, derivativesand mimetics of bacterial products thus produced will retain some or allof the antigenic activity possessed by a corresponding wild-typebacterial product to stimulate an effective host immune response (forexample, anti-LF antibody production). Thus, more effective bacterialvaccines and immunization methods are provided that yield sufficientstimulation of an anti-bacterial product (for example, anti-LF) immuneresponse in a subject, attended by diminished adverse side effectsassociated with nuclear hormone receptor modulation that would attendimmunization with the corresponding native bacterial product. Suchvariants, analogs and mimetics of bacterial products will exhibitsubstantially reduced or enhanced activity for repression or activationof one or more nuclear hormone receptor(s), and can, alternatively oradditionally, specifically inhibit, block, or enhance repression oractivation of one or more nuclear hormone receptor(s) by a correspondingnative bacterial product. For example, administration of a vaccineformulation that includes a modified anthrax LF analog or fragment willbe characterized by a reduction or increase in a level of GR and/or PRrepression, or in the occurrence of one or more inflammatory orautoimmune symptoms in the immunized subject, compared to that observedfollowing administration of a vaccine formulation comprising a similardose of native anthrax LF, of approximately 20%, 30%, 50%, 75% and up to95% or greater. At the same time, the vaccine formulation will elicit aneffective immune response (for example, anti-LF antibody production)that is at least 20%, 30%, 50%, 75% and up to 95% or greater in titer orintensity compared to an immune response stimulated by immunizationusing a similar dose of the corresponding native bacterial product.

The methods disclosed herein allow the production and selection ofanalogs, variants, derivatives and mimetics of LF and other bacterialproducts for generation of improved vaccines and other therapeuticformulations. According to the disclosure herein, these analogs,variants, derivatives and mimetics can be routinely generated, such asby creation of truncated fragments or recombinant variants having one ormore targeted amino acid substitutions, insertions or deletions. Foreach subject bacterial product contemplated herein, availablestructure-function data will be used to select candidate targets formodification within a native protein. For example, in the case ofanthrax LF, it is known that the approximately 90 kD protein plays animportant role in enhancing protective immunity. An inducible LFexpression system has been developed to generate recombinant LF suitablefor human vaccine trials (Singh et al., FEMS Microbiol. Lett. 209:301-5,2002. Generally known methods can be employed to generate recombinantforms of LF and to evaluate immunogenic and nuclear hormone modulatoractivities of the recombinant LF proteins for development of improvedvaccines. Specific targets for chemical modification and/or mutagenesisare also readily determined in accordance with the present disclosureand by reference to published structure-function data for subjectbacterial products. For example, the crystal structure of LF and itscomplex with the N terminus of MAPKK-2 has recently been published byPannifer et al. (Nature 414:229-33, 2001). LF comprises four domains:domain I binds the membrane-translocating component of anthrax toxin,the protective antigen (PA); domains II, m and IV together create a longdeep groove that holds a 16-residue N-terminal tail of MAPKK-2 beforecleavage. Domain II resembles the ADP-ribosylating toxin from Bacilluscereus, but the active site is divergent and serves to augment substraterecognition. Domain m is inserted into domain II, and reportedlyfeatures a duplicate structural element of domain II. Domain IV isdistantly related to the zinc metalloprotease family, and contains thecatalytic centre; it also resembles domain I. In one exemplaryembodiment, one or more of these domains, for example, domain IVimplicated in metalloprotease function, is deleted or mutated to yieldan increase or reduction in GR or PR repression activity accompanied byretention of substantial activity of the mutant LF as a prophylactic ortherapeutic immunogen. Additional embodiments utilize fusion proteins,conjugates and other analogs and derivatives of bacterial products asvaccine agents according to the above description (for example, seeMilne et al., Mol. Microbiol. 15:661-6, 1995, who describe chimericproteins composed of a PA recognition domain of LF (LFN; residues 1-255)fused to a heterologous protein segment). The purified fusion proteinsretained their functionality of complementing PA to mediatetranslocation of the fusion protein into cells in the presence of PA,and also retained ability to react with antisera against LF.

Analogs, variants, derivatives and mimetics of bacterial products foruse include natural or synthetic, therapeutically or prophylacticallyactive, peptides (comprised of two or more covalently linked aminoacids), proteins, peptide or protein fragments, peptide or proteinanalogs, peptide or protein mimetics, and chemically modifiedderivatives or salts of active peptides or proteins. Thus, as usedherein, the terms “analog” or “mimetic” of a bacterial product willoften be intended to embrace all of these active species, for example,peptides and proteins, peptide and protein fragments, peptide andprotein analogs, peptide and protein mimetics, peptide and proteinfusions and other conjugates, and chemically modified derivatives andsalts of active peptides or proteins. Often, the peptides or proteinsthat will find use in the methods disclosed herein are muteins that arereadily obtainable by partial substitution, addition, or deletion ofamino acids within a naturally occurring or native (for example,wild-type, naturally occurring mutant, or allelic variant) peptide orprotein sequence of a known bacterial product (for example, LF).Additionally, biologically active fragments of native peptides orproteins are included. Such mutant derivatives and fragments will oftensubstantially retain a desired biological activity of the native peptideor proteins. In the case of peptides or proteins having carbohydratechains, biologically active variants marked by alterations in thesecarbohydrate species are also included.

The peptides, proteins, analogs and mimetics for use within the methodsand compositions disclosed herein are often formulated in apharmaceutical composition comprising an effective amount of thepeptide, protein, analog or mimetic that will modulate activity of oneor more nuclear hormone receptors or alleviate one or more symptoms of abacterial infection, inflammatory disorder or autoimmune condition.

In additional embodiments, peptides or proteins for use can be modifiedby addition or conjugation of a synthetic polymer, such as polyethyleneglycol, a natural polymer, such as hyaluronic acid, or an optional sugar(for example galactose, mannose), sugar chain, or nonpeptide compound.Substances added to the peptide or protein by such modifications canspecify or enhance binding to certain receptors or antibodies orotherwise enhance intracellular delivery, activity, half-life, cell- ortissue-specific targeting, or other beneficial properties of the peptideor protein. For example, such modifications can render the peptide orprotein more lipophilic, such as may be achieved by addition orconjugation of a phospholipid or fatty acid. Further included within themethods and compositions disclosed herein are peptides and proteinsprepared by linkage (for example, chemical bonding) of two or morepeptides, protein fragments or functional domains (for example,extracellular, transmembrane and cytoplasmic domains, ligand-bindingregions, active site domains, immunogenic epitopes, and the like)—forexample fusion peptides and proteins recombinantly produced toincorporate the functional elements of a plurality of different peptidesor proteins in a single encoded molecule.

Biologically active peptides and proteins for use within the methods andcompositions disclosde herein include native or “wild-type” peptides andproteins and naturally occurring variants of these molecules, such asnaturally occurring allelic variants and mutant proteins. Also includedare synthetic, such as chemically or recombinantly engineered, peptidesand proteins, as well as peptide and protein “analogs” and chemicallymodified derivatives, fragments, conjugates, and polymers of naturallyoccurring peptides and proteins. As used herein, the term peptide orprotein “analog” is meant to include modified peptides and proteinsincorporating one or more amino acid substitutions, insertions,rearrangements or deletions as compared to a native amino acid sequenceof a selected peptide or protein, or of a binding domain, fragment,immunogenic epitope, or structural motif, of a selected peptide orprotein. Peptide and protein analogs thus modified will be selected forsubstantially conserved biological activity comparable to that of acorresponding native peptide or protein, or alternatively, reduced orincreased biological activity compared to activity exhibited by acorresponding native peptide or protein. For example, analogs, variants,derivatives and mimetics of bacterial products may be selected thatexhibit conserved, or substantially increased or decreased activity(compared to the wild-type peptide or protein) for specific binding toone or more nuclear hormone receptor cofactors, proteolytic activityagainst a nuclear hormone receptor cofactor or other substrate,modulatory activity of a nuclear hormone receptor, immunogenicity,and/or toxicity or activity for induction of inflammatory or autoimmuneresponses in a mammalian subject. In certain detailed aspects, analogs,variants, derivatives and mimetics of bacterial products are selectedthat exhibit approximately 20%, 30%, 50%, 85%, 95% or greater activitylevels compared to the corresponding native peptide or protein forspecific binding to one or more nuclear hormone receptor cofactors,proteolytic activity against a nuclear hormone receptor cofactor orother substrate, modulatory activity of a nuclear hormone receptor,immunogenicity, and/or toxicity or activity for induction ofinflammatory or autoimmune responses in a mammalian subject.

As disclosed herein, the term “biologically active peptide or proteinanalog” further includes derivatives or synthetic variants of a nativepeptide or protein, such as amino and/or carboxyl terminal deletions andfusions, as well as intrasequence insertions, substitutions or deletionsof single or multiple amino acids. Insertional amino acid sequencevariants are those in which one or more amino acid residues areintroduced into a predetermined site in the protein. Random insertion isalso possible with suitable screening of the resulting product.Deletional variants are characterized by removal of one or more aminoacids from the sequence. Substitutional amino acid variants are those inwhich at least one residue in the sequence has been removed and adifferent residue inserted in its place.

Where a native peptide or protein is modified by amino acidsubstitution, amino acids are generally replaced by other amino acidshaving similar, conservatively related chemical properties such ashydrophobicity, hydrophilicity, electronegativity, small or bulky sidechains, and the like. Residue positions which are not identical to thenative peptide or protein sequence are thus replaced by amino acidshaving similar chemical properties, such as charge or polarity, wheresuch changes are not likely to substantially effect the properties ofthe peptide or protein analog. These and other minor alterations willtypically substantially maintain biological properties of the modifiedpeptide or protein, including biological activity (such as binding to anadhesion molecule, or other ligand or receptor), immunoidentity (such asrecognition by one or more monoclonal antibodies that recognize a nativepeptide or protein), and other biological properties of thecorresponding native peptide or protein.

As used herein, the term “conservative amino acid substitution” refersto the general interchangeability of amino acid residues having similarside chains. For example, a commonly interchangeable group of aminoacids having aliphatic side chains is alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Examples of conservativesubstitutions include the substitution of a non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another.Likewise, the present disclosure contemplates the substitution of apolar (hydrophilic) residue such as between arginine and lysine, betweenglutamine and asparagine, and between threonine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another or the substitution of an acidicresidue such as aspartic acid or glutamic acid for another is alsocontemplated. Exemplary conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

The term “biologically active peptide or protein analog” furtherincludes modified forms of a native peptide or protein incorporatingstereoisomers (for example, D-amino acids) of the twenty conventionalamino acids, or unnatural amino acids such as α,α-disubstituted aminoacids, N-alkyl amino acids, lactic acid. These and other unconventionalamino acids may also be substituted or inserted within native peptidesand proteins useful within the methods and compositions disclosedherein. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and othersimilar amino acids and imino acids (for example, 4-hydroxyproline). Inaddition, biologically active peptide or protein analogs include singleor multiple substitutions, deletions and/or additions of carbohydrate,lipid and/or proteinaceous moieties that occur naturally or artificiallyas structural components of the subject peptide or protein, or are boundto or otherwise associated with the peptide or protein.

To facilitate production and use of peptide and protein analogs,reference can be made to molecular phylogenetic studies thatcharacterize conserved and divergent protein structural and functionalelements between different members of a species, genus, family or othertaxonomic group (such as between bacterial toxins of different species,or allelic or mutant variants of a toxin within a species). In thisregard, available studies will provide detailed assessments ofstructure-function relationships on a fine molecular level for modifyingthe majority of peptides and proteins disclosed herein to facilitateproduction and selection of operable peptide and protein analogs,including for a wide range of bacterial toxins and other bacterialproducts, as well as for cofactors and other agents involved inbacterial toxin-mediated modulation of nuclear hormone receptoractivity. These studies may include, for example, detailed sequencecomparisons identifying conserved and divergent structural elementsamong, for example, multiple isoforms or species or allelic variants ofa subject bacterial toxin (for example, LF, diptheria toxin, botulinumtoxin, or tetanus toxin) or multiple, related bacterial toxins. Suchconserved and divergent structural elements facilitate practice of themethods disclosed herein by pointing to useful targets for modifyingnative peptides and proteins to confer desired structural and/orfunctional changes.

In this context, existing sequence alignments may be analyzed andconventional sequence alignment methods may be employed to yieldsequence comparisons for analysis, for example to identify correspondingprotein regions and amino acid positions between protein family memberswithin a species, and between species variants of a protein of interest.These comparisons are useful to identify conserved and divergentstructural elements of interest, the latter of which will often beuseful for incorporation in a biologically active peptide or protein toyield a functional analog thereof. Typically, one or more amino acidresidues marking a divergent structural element of interest in adifferent reference peptide sequence is incorporated within thefunctional peptide or protein analog. For example, a cDNA encoding anative LF peptide or protein may be recombinantly modified at one ormore corresponding amino acid position(s) (for example, correspondingpositions that match or span a similar aligned sequence elementaccording to accepted alignment methods to residues marking thestructural element of interest in a heterologous reference peptide orprotein sequence, such as an isoform, species or allelic variant, orsynthetic mutant, of the subject LF peptide or protein) to encode anamino acid deletion, substitution, or insertion that alterscorresponding residue(s) in the native peptide or protein to generate anoperable peptide or protein analog of use—having an analogous structuraland/or functional element as the reference peptide or protein.

Within this rational design method for constructing biologically activepeptide and protein analogs, the native or wild-type identity ofresidue(s) at amino acid positions corresponding to a structural elementof interest in a heterologous reference peptide or protein may bealtered to the same, or a conservatively related, residue identity asthe corresponding amino acid residue(s) in the reference peptide orprotein. However, it is often possible to alter native amino acidresidues non-conservatively with respect to the corresponding referenceprotein residue(s). In particular, many non-conservative amino acidsubstitutions, particularly at divergent sites suggested to be moreamenable to modification, may yield a moderate impairment or neutraleffect, or even enhance a selected biological activity, compared to thefunction of a native peptide or protein.

Sequence alignment and comparisons to forecast useful peptide andprotein analogs and mimetics will be further refined by analysis ofcrystalline structure (see, for example, Löebermann et al., J. Molec.Biol. 177:531-556, 1984; Huber et al., Biochemistry 28:8951-8966, 1989;Stein et al., Nature 347:99-102, 1990; Wei et al., Structural Biology1:251-255, 1994, each incorporated herein by reference) of nativebiologically active proteins and peptides, coupled with computermodeling methods known in the art. These analyses allow detailedstructure-function mapping to identify desired structural elements andmodifications for incorporation into peptide and protein analogs andmimetics that will exhibit substantial activity comparable to that ofthe native peptide or protein for use within the methods andcompositions disclosed herein.

Biologically active peptide and protein analogs as disclosed hereintypically show substantial sequence identity to a corresponding nativepeptide or protein sequence. The term “substantial sequence identity”means that the two subject amino acid sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap penalties,share at least 65 percent sequence identity, commonly 80 percentsequence identity, often at least 90-95 percent or greater sequenceidentity. “Percentage amino acid identity” refers to a comparison of theamino acid sequences of two peptides or proteins which, when optimallyaligned, have approximately die designated percentage of the same aminoacids. Sequence comparisons are generally made to a reference sequenceover a comparison window of at least 10 residue positions, frequentlyover a window of at least 15-20 amino acids, wherein the percentage ofsequence identity is calculated by comparing a reference sequence to asecond sequence, the latter of which may represent, for example, apeptide analog sequence that includes one or more deletions,substitutions or additions which total 20 percent, typically less than5-10% of the reference sequence over the window of comparison. Thereference sequence may be a subset of a larger sequence, for example, asa segment of a LF protein. Optimal alignment of sequences for aligning acomparison window may be conducted according to the local homologyalgorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1981), by thehomology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), by the search for similarity method of Pearson and Lipman(Proc. Natl. Acad. Sci. USA 85:2444, 1988), or by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and/or TFASTA,such as provided in the Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, 575 Science Dr., Madison, Wis.).

By aligning a peptide or protein analog optimally with a correspondingnative peptide or protein, and by using appropriate assays, such asadhesion protein or receptor binding assays, to determine a selectedbiological activity, one can readily identify operable peptide andprotein analogs for use within the methods and compositions disclosedherein. Operable peptide and protein analogs are typically specificallyimmunoreactive with antibodies raised to the corresponding nativepeptide or protein. Likewise, nucleic acids encoding operable peptideand protein analogs will share substantial sequence identity asdescribed above to a nucleic acid encoding the corresponding nativepeptide or protein, and will typically selectively hybridize to apartial or complete nucleic acid sequence encoding the correspondingnative peptide or protein, or fragment thereof, under accepted, moderateor high stringency hybridization conditions (see, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual. 3^(rd) Edition, ColdSpring Harbor Laboratories, Cold Spring Harbor, N.Y., 2001, incorporatedherein by reference). The phrase “selectively hybridizing to” refers toa selective interaction between a nucleic acid probe that hybridizes,duplexes or binds preferentially to a particular target DNA or RNAsequence, for example when the target sequence is present in aheterogenous preparation such as total cellular DNA or RNA. Generally,nucleic acid sequences encoding biologically active peptide and proteinanalogs, or fragments thereof, will hybridize to nucleic acid sequencesencoding the corresponding native peptide or protein under stringentconditions (for example, selected to be about 5° C. lower than thethermal melting point (Tm) for the subject sequence at a defined ionicstrength and pH, where the Tm is the temperature under defined ionicstrength and pH at which 50% of the complementary or target sequencehybridizes to a perfectly matched probe). For discussions of nucleicacid probe design and annealing conditions, see, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual. 3rd Edition, Vols. 1-3,Cold Spring Harbor Laboratory, 2001 or Current Protocols in MolecularBiology, F. Ausubel et al, ed., Greene Publishing andWiley-Interscience, New York, 1987, each of which is incorporated hereinby reference. Typically, stringent or selective conditions will be thosein which the salt concentration is at least about 0.02 molar at pH 7 andthe temperature is at least about 60° C. Less stringent selectivehybridization conditions may also be chosen. As other factors maysignificantly affect the stringency of hybridization, including, amongothers, base composition and size of the complementary strands, thepresence of organic solvents and the extent of base mismatching, thecombination of parameters is more important than the specific measure ofany one.

Within additional embodiments, peptide mimetics are provided whichcomprise a peptide or non-peptide molecule that mimics the tertiarybinding structure and activity of a selected native peptide or proteinfunctional domain (for example, binding motif or active site). Thesepeptide mimetics include recombinantly or chemically modified peptides,as well as non-peptide agents such as small molecule drug mimetics, asfurther described below.

In one aspect, peptides (including polypeptides) of use are modified toproduce peptide mimetics by replacement of one or more naturallyoccurring side chains of the 20 genetically encoded amino acids (or Damino acids) with other side chains, for instance with groups such asalkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amidelower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy andthe lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-memberedheterocyclics. For example, proline analogs can be made in which thering size of the proline residue is changed from 5 members to 4, 6, or 7members. Cyclic groups can be saturated or unsaturated, and ifunsaturated, can be aromatic or non-aromatic. Heterocyclic groups cancontain one or more nitrogen, oxygen, and/or sulphur heteroatoms.Examples of such groups include the furazanyl, furyl, imidazolidinyl,imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (forexample morpholino), oxazolyl, piperazinyl (for example 1-piperazinyl),piperidyl (for example 1-piperidyl, piperidino), pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (for example 1-pyrrolidinyl), pyrrolinyl,pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (for examplethiomorpholino), and triazolyl. These heterocyclic groups can besubstituted or unsubstituted. Where a group is substituted, thesubstituent can be alkyl, alkoxy, halogen, oxygen, or substituted orunsubstituted phenyl.

Peptides and proteins, as well as peptide and protein analogs andmimetics, can also be covalently bound to one or more of a variety ofnonproteinaceous polymers, for example, polyethylene glycol,polypropylene glycol, or polyoxyalkenes, in the manner set forth in U.S.Pat. No. 4,640,835; U.S. Pat. No. 4,496,689; U.S. Pat. No. 4,301,144;U.S. Pat. No. 4,670,417; U.S. Pat. No. 4,791,192; or U.S. Pat. No.4,179,337, all which are incorporated by reference in their entiretyherein.

Other peptide and protein analogs and mimetics include glycosylationvariants, and covalent or aggregate conjugates with other chemicalmoieties. Covalent derivatives can be prepared by linkage offunctionalities to groups which are found in amino acid side chains orat the N- or C-termini, by means which are well known in the art. Thesederivatives can include, without limitation, aliphatic esters or amidesof the carboxyl terminus, or of residues containing carboxyl sidechains, O-acyl derivatives of hydroxyl group-containing residues, andN-acyl derivatives of the amino terminal amino acid or amino-groupcontaining residues, for example, lysine or arginine. Acyl groups areselected from the group of alkyl-moieties including C3 to C18 normalalkyl, thereby forming alkanoyl aroyl species. Covalent attachment tocarrier proteins, for example, immunogenic moieties can also beemployed.

In addition to these modifications, glycosylation alterations ofbiologically active peptides and proteins can be made, for example, bymodifying the glycosylation patterns of a peptide during its synthesisand processing, or in further processing steps. One means foraccomplishing this are by exposing the peptide to glycosylating enzymesderived from cells that normally provide such processing, for example,mammalian glycosylation enzymes. Deglycosylation enzymes can also besuccessfully employed to yield useful modified peptides and proteins.Also embraced are versions of a native primary amino acid sequence whichhave other minor modifications, including phosphorylated amino acidresidues, for example, phosphotyrosine, phosphoserine, orphosphothreonine, or other moieties, including ribosyl groups orcross-linking reagents.

Peptidomimetics may also have amino acid residues that have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those that havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets, for example, affinity ligands.

A major group of peptidomimetics comprises covalent conjugates of nativepeptides or proteins, or fragments thereof, with other proteins orpeptides. These derivatives can be synthesized in recombinant culturesuch as N- or C-terminal fusions or by the use of agents known in theart for their usefulness in cross-linking proteins through reactive sidegroups. Preferred peptide and protein derivatization sites for targetingby cross-linking agents are at free amino groups, carbohydrate moieties,and cysteine residues.

Fusion polypeptides between biologically active peptides or proteins andother homologous or heterologous peptides and proteins are alsoprovided. Many growth factors and cytokines are homodimeric entities,and a repeat construct of these molecules or active fragments thereofwill yield various advantages, including lessened susceptibility toproteolytic degradation. Repeat and other fusion constructs of bacterialproteins and peptides yield similar advantages within the methods andcompositions disclosed herein. Various alternative multimeric constructscomprising peptides and proteins of use are thus provided. In certainembodiments, biologically active polypeptide fusions are provided asdescribed in U.S. Pat. Nos. 6,018,026, 5,843,725, 6,291,646, 6,300,099,and 6,323,323 (each incorporated herein by reference), for example bylinking one or more biologically active peptides or proteins disclosedherein with a heterologous, multimerizing polypeptide or protein, forexample an immunoglobulin heavy chain constant region, or animmunoglobulin light chain constant region. The biologically active,multimerized polypeptide fusion thus constructed can be a hetero- orhomo-multimer, for example, a heterodimer or homodimer comprising one ormore bacterial proteins or peptides(s), which can each include one ormore distinct biologically active peptides or proteins operable withinthe methods and compositions disclosed herein. Other heterologouspolypeptides can be combined with the active peptide or protein to yieldfusions that exhibit a combination of properties or activities of thederivative proteins. Other typical examples are fusions of a reporterpolypeptide, for example, CAT or luciferase, with a peptide or proteinas described herein, to facilitate localization of the fused peptide orprotein (see, for example, Dull et al., U.S. Pat. No. 4,859,609,incorporated herein by reference). Other fusion partners useful in thiscontext include bacterial beta-galactosidase, trpE, Protein A,beta-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alphamating factor (see, for example, Godowski et al., Science 241:812-816,1988, incorporated herein by reference).

The use of biologically active peptides and proteins modified bycovalent or aggregative association with chemical moieties van also beused in the methods disclosed herein. These derivatives generally fallinto the three classes: (1) salts, (2) side chain and terminal residuecovalent modifications, and (3) adsorption complexes, for example withcell membranes. Such covalent or aggregative derivatives are useful forvarious purposes, for example as agonists or antagonists to nativebacterial products, as immunogens, as reagents in immunoassays, or inpurification methods such as for affinity purification of ligands orother binding ligands. For example, an active peptide or protein can beimmobilized by covalent bonding to a solid support such as cyanogenbromide-activated Sepharose, by methods which are well known in the art,or adsorbed onto polyolefin surfaces, with or without glutaraldehydecross-linking, for use in the assay or purification of antibodies thatspecifically bind the active peptide or protein. The active peptide orprotein can also be labeled with a detectable group, for exampleradioiodinated by the chloramine T procedure, covalently bound to rareearth chelates, or conjugated to another fluorescent moiety for use indiagnostic assays, including assays involving in vivo administration ofthe labeled peptide or protein to determine, such as nuclear hormonereceptor activity, succeptibility to a disease or condition associatedwith bacterial infection, or other related indicia.

Those of skill in the art recognize that a variety of techniques areavailable for constructing peptide and protein mimetics with the same,similar, increased, or reduced biological activity as the correspondingnative peptide or protein. Often these analogs, variants, derivativesand mimetics will exhibit one or more desired activities that aredistinct or improved from the corresponding native peptide or protein,for example improved characteristics of solubility, stability, and/orsusceptibility to hydrolysis or proteolysis (see, for example, Morganand Gainor, Ann. Rep. Med. Chem. 24:243-252, 1989, incorporated hereinby reference). Certain peptidomimetic compounds are based upon the aminoacid sequence of the proteins and peptides described herein, includingsequences of bacterial toxins such as LF. Typically, peptidomimeticcompounds are synthetic compounds having a three-dimensional structure(of at least part of the mimetic compound) that mimics, for example, theprimary, secondary, and/or tertiary structural, and/or electrochemicalcharacteristics of a selected peptide or protein, or a structuraldomain, active site, or binding region (for example, a homotypic orheterotypic binding site, catalytic active site or domain, receptor orligand binding interface or domain, etc.) thereof. The peptide-mimeticstructure or partial structure (also referred to as a peptidomimetic“motif” of a peptidomimetic compound) will often share a desiredbiological activity with a native peptide or protein, as discussed above(for example, receptor or cofactor binding and/or activation orrepression activities, immunogenic activity (such as binding to MHCmolecules of one or multiple haplotypes and activating CD8⁺ and/or CD4⁺T), etc. Typically, at least one subject biological activity of themimetic compound is not substantially reduced in comparison to, and isoften the same as or greater than, the activity of the native peptide onwhich the mimetic was modeled. In addition, peptidomimetic compounds canhave other desired characteristics that enhance their therapeuticapplication, such as increased cell permeability, greater affinityand/or avidity, and prolonged biological half-life. The peptidomimeticswill sometimes have a “backbone” that is partially or completelynon-peptide, but with side groups identical to the side groups of theamino acid residues that occur in the peptide or protein on which thepeptidomimetic is modeled. Several types of chemical bonds, for exampleester, thioester, thioamide, retroamide, reduced carbonyl, dimethyleneand ketomethylene bonds, are known in the art to be generally usefulsubstitutes for peptide bonds in the construction of protease-resistantpeptidomimetics.

The following describes methods for preparing peptide and proteinmimetics modified at the N-terminal amino group, the C-terminal carboxylgroup, and/or changing one or more of the amido linkages in the peptideto a non-amido linkage. It being understood that two or more suchmodifications can be coupled in one peptide or protein mimetic structure(for example, modification at the C-terminal carboxyl group andinclusion of a —CH₂-carbamate linkage between two amino acids in thepeptide. For N-terminal modifications, peptides typically aresynthesized as the free acid but, as noted above, can be readilyprepared as the amide or ester. One can also modify the amino and/orcarboxy terminus of peptide compounds to produce other compounds of use.Amino terminus modifications include methylating (for example, —NHCH₃ or—NH(CH₃)₂), acetylating, adding a carbobenzoyl group, or blocking theamino terminus with any blocking group containing a carboxylatefunctionality defined by RCOO—, where R is selected from the groupconsisting of naphthyl, acridinyl, steroidyl, and similar groups.Carboxy terminus modifications include replacing the free acid with acarboxamide group or forming a cyclic lactam at the carboxy terminus tointroduce structural constraints. Amino terminus modifications are asrecited above and include alkylating, acetylating, adding a carbobenzoylgroup, forming a succinimide group, etc. The N-terminal amino group canthen be reacted as follows:

-   -   (a) to form an amide group of the formula RC(O)NH— where R is as        defined above by reaction with an acid halide [for example,        RC(O)Cl] or acid anhydride. Typically, the reaction can be        conducted by contacting about equimolar or excess amounts (for        example, about 5 equivalents) of an acid halide to the peptide        in an inert diluent (for example, dichloromethane) preferably        containing an excess (for example, about 10 equivalents) of a        tertiary amine, such as diisopropylethylamine, to scavenge the        acid generated during reaction. Reaction conditions are        otherwise conventional (for example, room temperature for 30        minutes). Alkylation of the terminal amino to provide for a        lower alkyl N-substitution followed by reaction with an acid        halide as described above will provide for N-alkyl amide group        of the formula RC(O)NR—;    -   (b) to form a succinimide group by reaction with succinic        anhydride. As before, an approximately equimolar amount or an        excess of succinic anhydride (for example, about 5 equivalents)        can be employed and the amino group is converted to the        succinimide by methods well known in the art including the use        of an excess (for example, ten equivalents) of a tertiary amine        such as diisopropylethylamine in a suitable inert solvent (for        example, dichloromethane) (see, for example, Wollenberg, et al.,        U.S. Pat. No. 4,612,132, incorporated herein by reference). It        is understood that the succinic group can be substituted with,        for example, C₂-C₆ alky or —SR substituents that are prepared in        a conventional manner to provide for substituted succinimide at        the N-terminus of the peptide. Such alkyl substituents are        prepared by reaction of a lower olefin (C₂-C₆) with maleic        anhydride in the manner described by Wollenberg, et al. (U.S.        Pat. No. 4,612,132) and —SR substituents are prepared by        reaction of RSH with maleic anhydride where R is as defined        above;    -   (c) to form a benzyloxycarbonyl-NH— or a substituted        benzyloxycarbonyl-NH— group by reaction with approximately an        equivalent amount or an excess of CBZ-CL (for example,        benzyloxycarbonyl chloride) or a substituted CBZ-Cl in a        suitable inert diluent (for example, dichloromethane) preferably        containing a tertiary amine to scavenge the acid generated        during the reaction;    -   (d) to form a sulfonamide group by reaction with an equivalent        amount or an excess (for example, 5 equivalents) of R—S(O)₂Cl in        a suitable inert diluent (dichloromethane) to convert the        terminal amine into a sulfonamide where R is as defined above.        Preferably, the inert diluent contains excess tertiary amine        (for example, ten equivalents) such as diisopropylethylamine, to        scavenge the acid generated during reaction. Reaction conditions        are otherwise conventional (for example, room temperature for 30        minutes);    -   (e) to form a carbamate group by reaction with an equivalent        amount or an excess (for example, 5 equivalents) of R—OC(O)Cl or        R—OC(O)OC₆H₄-p-NO₂ in a suitable inert diluent (for example,        dichloromethane) to convert the terminal amine into a carbamate        where R is as defined above. Preferably, the inert diluent        contains an excess (for example, about 10 equivalents) of a        tertiary amine, such as diisopropylethylamine, to scavenge any        acid generated during reaction. Reaction conditions are        otherwise conventional (for example, room temperature for 30        minutes);    -   (f) to form a urea group by reaction with an equivalent amount        or an excess (for example, 5 equivalents) of R—N═C═O in a        suitable inert diluent (for example, dichloromethane) to convert        the terminal amine into a urea (for example, RNHC(O)NH—) group        where R is as defined above. Preferably, the inert diluent        contains an excess (for example, about 10 equivalents) of a        tertiary amine, such as diisopropylethylamine. Reaction        conditions are otherwise conventional (for example, room        temperature for about 30 minutes).

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by an ester (for example, —C(O)OR where R is as defined above),resins as used to prepare peptide acids are typically employed, and theside chain protected peptide is cleaved with base and the appropriatealcohol, for example, methanol. Side chain protecting groups are thenremoved in the usual fashion by treatment with hydrogen fluoride toobtain the desired ester.

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by the amide —C(O)NR₃R₄, a benzhydrylamine resin is used as thesolid support for peptide synthesis. Upon completion of the synthesis,hydrogen fluoride treatment to release the peptide from the supportresults directly in the free peptide amide (for example, the C-terminusis —C(O)NH₂). Alternatively, use of the chloromethylated resin duringpeptide synthesis coupled with reaction with ammonia to cleave the sidechain protected peptide from the support yields the free peptide amideand reaction with an alkylamine or a dialkylamine yields a side chainprotected alkylamide or dialkylamide (for example, the C-terminus is—C(O)NRR₁ where R and R₁ are as defined above). Side chain protection isthen removed in the usual fashion by treatment with hydrogen fluoride togive the free amides, alkylamides, or dialkylamides.

In another alternative embodiments, the C-terminal carboxyl group or aC-terminal ester of a biologically active peptide can be induced tocyclize by internal displacement of the —OH or the ester (—OR) of thecarboxyl group or ester respectively with the N-terminal amino group toform a cyclic peptide. For example, after synthesis and cleavage to givethe peptide acid, the free acid is converted to an activated ester by anappropriate carboxyl group activator such as dicyclohexylcarbodiimide(DCC) in solution, for example, in methylene chloride (CH₂Cl₂), dimethylformamide (DMF) mixtures. The cyclic peptide is then formed by internaldisplacement of the activated ester with the N-terminal amine. Internalcyclization as opposed to polymerization can be enhanced by use of verydilute solutions. Such methods are well known in the art.

One can cyclize active peptides for use, or incorporate a desamino ordescarboxy residue at the termini of the peptide, so that there is noterminal amino or carboxyl group, to decrease susceptibility toproteases, or to restrict the conformation of the peptide. C-terminalfunctional groups among peptide analogs and mimetics include amide,amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, andcarboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

Other methods for making peptide and protein derivatives and mimeticsfor use within the methods and compositions disclosed herein aredescribed in Hruby et al. (Biochem J. 268(2:249-262, 1990, incorporatedherein by reference). According to these methods, biologically activepeptides and proteins serve as structural models for non-peptide mimeticcompounds having similar biological activity as the native peptide orprotein. Those of skill in the art recognize that a variety oftechniques are available for constructing compounds with the same orsimilar desired biological activity as the lead peptide or proteincompound, or that have more favorable activity than the lead withrespect a desired property such as solubility, stability, andsusceptibility to hydrolysis and proteolysis (see, for example, Morganand Gainor, Ann. Rep. Med. Chem. 24:243-252, 1989, incorporated hereinby reference). These techniques include, for example, replacing apeptide backbone with a backbone composed of phosphonates, amidates,carbamates, sulfonamides, secondary amines, and/or N-methylamino acids.

Peptide and protein mimetics wherein one or more of the peptidyllinkages [—C(O)NH—] have been replaced by such linkages as a—CH₂-carbamate linkage, a phosphonate linkage, a —CH₂-sulfonamidelinkage, a urea linkage, a secondary amine (—CH₂NH—) linkage, and analkylated peptidyl linkage [—C(O)NR₆— where R₆ is lower alkyl] areprepared, for example, during conventional peptide synthesis by merelysubstituting a suitably protected amino acid analogue for the amino acidreagent at the appropriate point during synthesis. Suitable reagentsinclude, for example, amino acid analogues wherein the carboxyl group ofthe amino acid has been replaced with a moiety suitable for forming oneof the above linkages. For example, if one desires to replace a —C(O)NR—linkage in the peptide with a —CH₂-carbamate linkage (—CH₂OC(O)NR—),then the carboxyl (—COOH) group of a suitably protected amino acid isfirst reduced to the —CH₂OH group which is then converted byconventional methods to a —OC(O)Cl functionality or apara-nitrocarbonate —OC(O)O—C₆H₄-p-NO₂ functionality. Reaction of eitherof such functional groups with the free amine or an alkylated amine onthe N-terminus of the partially fabricated peptide found on the solidsupport leads to the formation of a —CH₂OC(O)NR— linkage. For a moredetailed description of the formation of such —CH₂-carbamate linkages,see, for example, Cho et al. (Science 261:1303-1305, 1993, incorporatedherein by reference).

Replacement of an amido linkage in an active peptide with a—CH₂-sulfonamide linkage can be achieved by reducing the carboxyl(—COOH) group of a suitably protected amino acid to the —CH₂OH group,and the hydroxyl group is then converted to a suitable leaving groupsuch as a tosyl group by conventional methods. Reaction of thederivative with, for example, thioacetic acid followed by hydrolysis andoxidative chlorination will provide for the —CH₂—S(O)₂Cl functionalgroup which replaces the carboxyl group of the otherwise suitablyprotected amino acid. Use of this suitably protected amino acid analoguein peptide synthesis provides for inclusion of an —CH₂S(O)₂NR— linkagethat replaces the amido linkage in the peptide thereby providing apeptide mimetic. For a more complete description on the conversion ofthe carboxyl group of the amino acid to a —CH₂S(O)₂Cl group, see, forexample, Weinstein and Boris (Chemistry & Biochemistry of Amino Acids.Peptides and Proteins, Vol. 7, pp. 267-357, Marcel Dekker, Inc., NewYork, 1983, incorporated herein by reference). Replacement of an amidolinkage in an active peptide with a urea linkage can be achieved, forexample, in the manner set forth in U.S. patent application Ser. No.08/147,805 (incorporated herein by reference).

Secondary amine linkages wherein a—CH₂NH— linkage replaces the amidolinkage in the peptide can be prepared by employing, for example, asuitably protected dipeptide analogue wherein the carbonyl bond of theamido linkage has been reduced to a CH₂ group by conventional methods.For example, in the case of diglycine, reduction of the amide to theamine will yield after deprotection H₂NCH₂CH₂NHCH₂ COOH that is thenused in N-protected form in the next coupling reaction. The preparationof such analogues by reduction of the carbonyl group of the amidolinkage in the dipeptide is well known in the art.

The biologically active peptide and protein agents of the presentdisclosure can exist in a monomeric form with no disulfide bond formedwith the thiol groups of cysteine residue(s) that may be present in thesubject peptide or protein. Alternatively, an intermolecular disulfidebond between thiol groups of cysteines on two or more peptides orproteins can be produced to yield a multimeric (for example, dimeric,tetrameric or higher oligomeric) compound. Certain of such peptides andproteins can be cyclized or dimerized via displacement of the leavinggroup by the sulfur of a cysteine or homocysteine residue (see, forexample, Barker et al., J. Med. Chem. 35:2040-2048, 1992; and Or et al.,J. Org. Chem. 56:3146-3149, 1991, each incorporated herein byreference). Thus, one or more native cysteine residues may besubstituted with a homocysteine. Intramolecular or intermoleculardisulfide derivatives of active peptides and proteins provide analogs inwhich one of the sulfurs has been replaced by a CH₂ group or otherisostere for sulfur. These analogs can be made via an intramolecular orintermolecular displacement, using methods known in the art.

Within certain embodiments, delivery of biologically active agents,including native bacterial products and analogs, variants, derivativesand mimetics thereof, is enhanced by methods and agents that targetselective transport mechanisms and promote endo- or transcytocis ofmacromoloecular drugs. In this regard, the compositions and deliverymethods optionally incorporate a selective transport-enhancing agentthat facilitates transport of one or more biologically active agents.These transport-enhancing agents can be employed in a combinatorialformulation or coordinate administration protocol with one or more ofthe peptides, proteins, analogs and mimetics disclosed herein, tocoordinately enhance delivery of the biologically active agent(s) intotarget cells. Exemplary selective transport-enhancing agents for usewithin this aspect include, but are not limited to, glycosides,sugar-containing molecules, and binding agents such as lectin bindingagents, which are known to interact specifically with epithelialtransport barrier components (see, for example, Goldstein et al., Annu.Rev. Cell. Biol. 1:1-39, 1985, incorporated herein by reference). Forexample, specific “bioadhesive” ligands, including various plant andbacterial lectins, which bind to cell surface sugar moieties byreceptor-mediated interactions can be employed as carriers or conjugatedtransport mediators for enhancing delivery of biologically activeagents. Certain bioadhesive ligands of use will mediate transmission ofbiological signals to epithelial target cells that trigger selectiveuptake of the adhesive ligand by specialized cellular transportprocesses (endocytosis or transcytosis). These transport mediators cantherefore be employed as a “carrier system” to stimulate or directselective uptake of one or more biologically active agent(s) within themethods disclosed herein. To utilize these transport-enhancing agents,general carrier formulation and/or conjugation methods known in the artare used to coordinately administer a selective transport enhancer (forexample, a receptor-specific ligand) and a biologically active agent toa subject to trigger or mediate enhanced endo- or transcytosis of theactive agent into specific target cell(s), tissue(s) or compartment(s).

“Lectins” are plant proteins that bind to specific sugars found on thesurface of glycoproteins and glycolipids of eukaryotic cells.Concentrated solutions of lectins have a ‘mucotractive’ effect, andvarious studies have demonstrated rapid receptor mediated endocytocis(RME) of lectins and lectin conjugates (for example, concanavalin Aconjugated with colloidal gold particles) across mucosal surfaces.Additional studies have reported that the uptake mechanisms for lectinscan be utilized for intestinal drug targeting in vivo. In certain ofthese studies, polystyrene nanoparticles (500 nm) were covalentlycoupled to tomato lectin and reported yielded improved systemic uptakeafter oral administration to rats.

In addition to plant lectins, microbial adhesion and invasion factorsprovide a rich source of candidates for use as adhesive/selectivetransport carriers within the compositions and methods disclosed herein(see, for example, Lehr, Crit. Rev. Therap. Drug Carrier Syst.11:177-218, 1995; Swann, Pa., Pharmaceutical Research 15:826-832, 1998,each incorporated herein by reference). Two components are necessary forbacterial adherence processes, a bacterial ‘adhesin’ (adherence orcolonization factor) and a receptor on the host cell surface. Bacteriacausing mucosal infections need to penetrate the mucus layer beforeattaching themselves to the epithelial surface. This attachment isusually mediated by bacterial fimbriae or pilus structures, althoughother cell surface components may also take part in the process.Adherent bacteria colonize mucosal epithelia by multiplication andinitiation of a series of biochemical reactions inside the target cellthrough signal transduction mechanisms (with or without the help oftoxins). Associated with these invasive mechanisms, a wide diversity ofbioadhesive proteins (for example, invasin, internalin) originallyproduced by various bacteria and viruses are known. These allow forextracellular attachment of such microorganisms with an impressiveselectivity for host species and even particular target tissues. Signalstransmitted by such receptor-ligand interactions trigger the transportof intact, living microorganisms into, and eventually through,epithelial cells by endo- and transcytotic processes. Such naturallyoccurring phenomena may be harnessed (for example, by complexingbiologically active agents such as bacterial toxin with an adhesin)according to the teachings herein for enhanced delivery of biologicallyactive compounds to target sites of drug action. One advantage of thisstrategy is that the selective carrier partners thus employed aresubstrate-specific, leaving the natural barrier function of epithelialtissues intact against other solutes (see, for example, Lehr, DrugAbsorption Enhancement, pp. 325-362, de Boer, Ed., Harwood AcademicPublishers, 1994, incorporated herein by reference).

Various bacterial and plant toxins that bind epithelial surfaces in aspecific, lectin-like manner are also useful within the methods andcompositions disclosed herein. For example, diptheria toxin (DT) entershost cells rapidly by RME. Likewise, the B subunit of the E. coli heatlabile toxin binds to the brush border of intestinal epithelial cells ina highly specific, lectin-like manner. Uptake of this toxin andtranscytosis to the basolateral side of the enterocytes has beenreported in vivo and in vitro. Other researches have expressed thetransmembrane domain of diphtheria toxin in E. coli as a maltose-bindingfusion protein and coupled it chemically to high-Mw poly-L-lysine. Theresulting complex was successfully used to mediate internalization of areporter gene in vitro. In addition to these examples, Staphylococcusaureus produces a set of proteins (for example, staphylococcalenterotoxin A (SEA), SEB, toxic shock syndrome toxin 1 (TSST-1) whichact both as superantigens and toxins. Studies relating to these proteinshave reported dose-dependent, facilitated transcytosis of SEB and TSST-Iin Caco-2 cells.

Various plant toxins, mostly ribosome-inactivating proteins (RIPs), havebeen identified that bind to any mammalian cell surface expressinggalactose units and are subsequently internalized by RME. Toxins such asnigrin b, α-sarcin, ricin and saporin, viscumin, and modeccin are highlytoxic upon oral administration (for example, are rapidly internalized).Therefore, modified, less toxic subunits of these compound will beuseful to facilitate the uptake of biologically active agents, includingbacterial products and analogs, variants, derivatives and mimeticsthereof.

Viral haemagglutinins comprise another type of transport agent tofacilitate delivery of biologically active agents within the methods andcompositions disclosed herein. The initial step in many viral infectionsis the binding of surface proteins (haemagglutinins) to mucosal cells.These binding proteins have been identified for most viruses, includingrotaviruses, varicella zoster virus, semliki forest virus, adenoviruses,potato leafroll virus, and reovirus. These and other exemplary viralhemagglutinins can be employed in a combinatorial formulation (forexample, a mixture or conjugate formulation) or coordinateadministration protocol with, for example, one or more bacterialproducts or analogs, variants, derivatives and mimetics thereof.Alternatively, viral hemagglutinins can be employed in a combinatorialformulation or coordinate administration protocol to directly enhancedelivery of a biologically active agent.

A variety of endogenous, selective transport-mediating factors are alsoavailable for use within the methods and compositions disclosed herein.Mammalian cells have developed an assortment of mechanisms to facilitatethe internalization of specific substrates and target these to definedcompartments. Collectively, these processes of membrane deformations aretermed ‘endocytosis’ and comprise phagocytosis, pinocytosis,receptor-mediated endocytosis (clathrin-mediated RME), and potocytosis(non-clathrin-mediated RME). RME is a highly specific cellular biologicprocess by which, as its name implies, various ligands bind to cellsurface receptors and are subsequently internalized and traffickedwithin the cell. In many cells the process of endocytosis is so activethat the entire membrane surface is internalized and replaced in lessthan a half hour.

RME is initiated when specific ligands bind externally oriented membranereceptors. Binding occurs quickly and is followed by membraneinvagination until an internal vesicle forms within the cell (the earlyendosome, “receptosome”, or CURL (compartment of uncoupling receptor andligand). Localized membrane proteins, lipids and extracellular solutesare also internalized during this process. When the ligand binds to itsspecific receptor, the ligand-receptor complex accumulates in coatedpits. Coated pits are areas of the membrane with high concentration ofendocellular clathrin subunits. The assembly of clathrin molecules onthe coated pit is believed to aid the invagination process. Specializedcoat proteins called adaptins, trap specific membrane receptors thatmove laterally through the membrane in the coated pit area by binding toa signal sequence (Tyr-X-Arg-Phe, where X=any amino acid) at theendocellular carboxy terminus of the receptor. This process ensures thatthe correct receptors are concentrated in the coated pit areas andminimizes the amount of extracellular fluid that is taken up in thecell.

Following the internalization process, the clathrin coat is lost throughthe help of chaperone proteins, and proton pumps lower the endosomal pHto approximately 5.5, which causes dissociation of the receptor-ligandcomplex. CURL serves as a compartment to segregate the recyclingreceptor (for example transferrin) from receptor involved intranscytosis (for example transcoba-lamin). Endosomes may then moverandomly or by saltatory motion along the microtubules until they reachthe trans-Golgi reticulum where they are believed to fuse with Golgicomponents or other membranous compartments and convert intotubulovesicular complexes and late endosomes or multivesicular bodies.The fate of the receptor and ligand are determined in these sortingvesicles. Some ligands and receptors are returned to the cell surfacewhere the ligand is released into the extracellular milieu and thereceptor is recycled. Alternatively, the ligand is directed to lysosomesfor destruction while the receptor is recycled to the cell membrane. Theendocytotic recycling pathways of polarized epithelial cells aregenerally more complex than in non-polarized cells. In these enterocytesa common recycling compartment exists that receives molecules from bothapical and basolateral membranes and is able to correctly return them tothe appropriate membrane or membrane recycling compartment

Current understanding of RME receptor structure and relatedstructure-function relationships has been significantly enhanced by thecloning of mRNA sequences coding for endocytotic receptors. Most RMEreceptors share principal structural features, such as an extracellularligand binding site, a single hydrophobic transmembrane domain (unlessthe receptor is expressed as a dimer), and a cytoplasmic tail encodingendocytosis and other functional signals. Two classes of receptors areproposed based on their orientation in the cell membrane; the aminoterminus of Type I receptors is located on the extracellular side of themembrane, whereas Type II receptors have this same protein tail in theintracellular milieu.

As noted above, potocytosis, or non-clathrin coated endocytosis, takesplace through caveolae, which are uniform omega- or flask-shapedmembrane invaginations 50-80 nm in diameter. This process was firstdescribed as the internalization mechanism of the vitamin folic acid.Morphological studies have implicated caveolae in i) the transcytosis ofmacromolecules across endothelial cells; (ii) the uptake of smallmolecules via potocytosis involving GPI-linked receptor molecules and anunknown anion transport protein; iii) interactions with the actin-basedcytoskeleton; and (iv) the compartmentalization of certain signalingmolecules involved in signal transduction, including G-protein coupledreceptors. Caveolae are characterized by the presence of an integral22-kDa membrane protein termed VIP21-caveolin, which coats thecytoplasmic surface of the membrane. From a drug delivery standpoint,the advantage of potocytosis pathways over clathrin-coated RME pathwayslies in the absence of the pH lowering step, which circumvents theendosomal/lysosomal pathway. This pathway for selectivetransporter-mediated delivery of biologically active agents is thereforeparticularly effective for enhanced delivery of pH-sensitivemacromolecules.

Exemplary among potocytotic transport carriers mechanisms for use is thefolate carrier system, which mediates transport of the vitamin folicacid (FA) into target cells via specific binding to the folate receptor(FR) (see, for example, Reddy et al., Crit. Rev. Ther. Drug Car. Syst.15:587-627, 1998, incorporated herein by reference). The cellular uptakeof free folic acid is mediated by the folate receptor and/or the reducedfolate carrier. The folate receptor is a glycosylphosphatidylinositol(GPI)-anchored 38 kDa glycoprotein clustered in caveolae mediating celltransport by potocytosis. While the expression of the reduced folatecarrier is ubiquitously distributed in eukaryotic cells, the folatereceptor is principally overexpressed in human tumors. Two homologousisoforms (α and β) of the receptor have been identified in humans. Theα-isoform is found to be frequently overexprssed in epithelial tumors,whereas the β-form is often found in non-epithelial lineage tumors.Consequently, this receptor system has been used in drug-targetingapproaches to cancer cells, but also in protein delivery, gene delivery,and targeting of antisense oligonucleotides to a variety of cell types.

Folate-drug conjugates are well suited for use within the methods andcompositions disclosed herein, because they allow penetration of targetcells exclusively via FR-mediated endocytosis. When FA is covalentlylinked, for example, via its γ-carboxyl to a biologically active agent,FR binding affinity (KD˜10⁻¹⁰M) is not significantly compromised, andendocytosis proceeds relatively unhindered, promoting uptake of theattached active agent by the FR-expressing cell. Because FRs aresignificantly overexpressed on a large fraction of human cancer cells(for example, ovarian, lung, breast, endometrial, renal, colon, andcancers of myeloid hematopoietic cells), this methodology allows forselective delivery of a wide range of therapeutic as well as diagnosticagents to tumors. Folate-mediated tumor targeting has been exploited todate for delivery of the following classes of molecules and molecularcomplexes: (i) protein toxins, (ii) low-molecular-weightchemotherapeutic agents, (iii) radioimaging agents, (iv) MRI contrastagents, (v) radio-therapeutic agents, (vi) liposomes with entrappeddrugs, (vii) genes, (viii) antisense oligonucleotides, (ix) ribozymes,and (x) immunotherapeutic agents (see, for example, Swann, Pa.,Pharmaceutical Research 15:826-832, 1998, incorporated herein byreference). In virtually all cases, in vitro studies demonstrate asignificant improvement in potency and/or cancer-cell specificity overthe nontargeted form of the same pharmaceutical agent.

In addition to the folate receptor pathway, a variety of additionalmethods to stimulate transcytosis within the disclosed methods aredirected to the transferrin receptor pathway, and the riboflavinreceptor pathway. In one aspect, conjugation of a biologically activeagent to riboflavin can effectuate RME-mediated uptake. Yet additionalembodiments utilize vitamin B12 (cobalamin) as a specialized transportprotein (for example, conjugation partner) to facilitate entry ofbiologically active agents into target cells. Certain studies suggestthat this particular system can be employed for the intestinal uptake ofluteinizing hormone releasing factor (LHRH)-analogs, granulocyte colonystimulating factor (G-CSF, 18.8 kDa), erythropoietin (29.5 kDa),α-interferon, and the LHRH-antagonist ANTIDE.

Still other embodiments utilize transferrin as a carrier or stimulant ofRME of mucosally delivered biologically active agents. Transferrin, an80 kDa iron-transporting glycoprotein, is efficiently taken up intocells by RME. Transferrin receptors are found on the surface of mostproliferating cells, in elevated numbers on erythroblasts and on manykinds of tumors. According to current knowledge of intestinal ironabsorption, transferrin is excreted into the intestinal lumen in theform of apotransferrin and is highly stable to attacks from intestinalpeptidases. In most cells, diferric transferrin binds to transferrinreceptor (TfR), a dimeric transmembrane glycoprotein of 180 kDa, and theligand-receptor complex is endocytosed within clathrin-coated vesicles.After acidification of these vesicles, iron dissociates from thetransferrin/TfR complex and enters the cytoplasm, where it is bound byferritin (Fn). Recent reports suggest that insulin covalently coupled totransferrin, is transported across Cac6-2 cell monolayers by RME. Otherstudies suggest that oral administration of this complex tostreptozotocin-induced diabetic mice significantly reduces plasmaglucose levels (28%), which is further potentiated by BFA pretreatmentThe transcytosis of transferrin (Tf) and transferrin conjugates isreportedly enhanced in the presence of Brefeldin A (BFA), a fungalmetabolite. In other studies, BFA treatment has been reported to rapidlyincrease apical endocytosis of both ricin and HRP in MDCK cells. Thus,BFA and other agents that stimulate receptor-mediated transport can beemployed within the methods disclosed herein as combinatoriallyformulated (for example, conjugated) and/or coordinately administeredagents to enhance receptor-mediated transport of biologically activeagents, including, for example, bacterial toxins and analogs, variants,derivatives and mimetics thereof.

Immunoglobulin transport mechanisms provide yet additional endogenouspathways and reagents for incorporation within the methods andcompositions disclosed herein. Receptor-mediated transcytosis ofimmunoglobulin G (IgG) across the neonatal small intestine serves toconvey passive immunity to many newborn mammals. In rats, IgG in milkselectively binds to neonatal Fc receptors (FcRn) expressed on thesurface of the proximal small intestinal enterocytes during the firstthree weeks after birth. FcRn binds IgG in a pH-dependent manner, withbinding occurring at the luminal pH (approx. 6-6.5) of the jejunum andrelease at the pH of plasma (approx. 7.4). The Fc receptor resembles themajor histocompatibility complex (MHC) class I antigens in that itconsists of two subunits, a transmembrane glycoprotein (gp50) inassociation with β2-microglobulin. In mature absorptive cells bothsubunits are colocalized in each of the membrane compartments thatmediate transcytosis of IgG. IgG administered in situ apparently causesboth subunits to concentrate within endocytic pits of the apical plasmamembrane, suggesting that ligand causes redistribution of receptors atthis site. These results support a model for transport in which IgG istransferred across the cell as a complex with both subunits.

Within the methods and compositions disclosed herein, IgG and otherimmune system-related carriers (including polyclonal and monoclonalantibodies and various fragments thereof) can be coordinatelyadministered with biologically active agents to provide for targeteddelivery, typically by receptor-mediated transport, of the biologicallyactive agent. For example, the biologically active agent may becovalently linked to the IgG or other immunological active agent or,alternatively, formulated in liposomes or other carrier vehicle which isin turn modified (such as coated or covalently linked) to incorporateIgG or other immunological transport enhancer. In certain embodiments,polymeric IgA and/or IgM transport agents are employed, which bind tothe polymeric immunoglobulin receptors (pIgRs) of target epithelialcells. Within these methods, expression of pIgR can be enhanced bycytokines.

Within other embodiments, antibodies and other immunological transportagents can themselves be modified for enhanced delivery of biologicallyactive agents. For example, antibodies can be more effectivelyadministered by charge modifying techniques. In one such aspect, anantibody drug delivery strategy involving antibody cationization isutilized that facilitates both trans-endothelial migration and targetcell endocytosis (see, for example, Pardridge, et al., JPET 286:548-544,1998, incorporated herein by reference). In one such strategy, the pI ofthe antibody is increased by converting surface carboxyl groups of theprotein to extended primary amino groups. These canonized homologousproteins have no measurable tissue toxicity and have minimalimmunogenicity. In addition, monoclonal antibodies may be cationizedwith retention of affinity for the target protein.

Additional selective transport-enhancing agents for use within themethods disclosed herein comprise whole bacteria and viruses, includinggenetically engineered bacteria and viruses, as well as components ofsuch bacteria and viruses. In addition to conventional gene deliveryvectors (for example, adenovirus) and related methods, this aspectincludes the use of bacterial ghosts and subunit constructs, forexample, as described by Huter et al., Journal of Controlled Release61:51-63, 1999 (incorporated herein by reference). Bacterial ghosts arenon-denatured bacterial cell envelopes, for example as produced by thecontrolled expression of the plasmid-encoded lysis gene E ofbacteriophage PhiXI74 in gram-negative bacteria. Protein E-specificlysis does not cause any physical or chemical denaturation to bacterialsurface structures, and bacterial ghosts are therefore useful indevelopment of inactivated whole-cell vaccines. Ghosts produced fromActinobacillus pleuropneumoniae, Pasteurella haemolytica and Salmonellasp. have proved successful in vaccination experiments. Recombinantbacterial ghosts can be created by the expression of foreign genes fusedto a membrane-targeting sequence, and thus can carry foreign therapeuticpeptides and proteins anchored in their envelope. The fact thatbacterial ghosts preserve a native cell wall, including bioadhesivestructures like fimbriae of their living counterparts, makes themsuitable for the attachment to specific target tissues such as mucosalsurfaces. Bacterial ghosts have been shown to be readily taken up bymacrophages, thus adhesion of ghosts to specific tissues can be followedby uptake through phagocytes.

In view of the foregoing, a wide variety of ligands involved inreceptor-mediated transport mechanisms are known in the art and can bevariously employed within the methods and compositions disclosed herein(for example, as conjugate partners or coordinately administeredmediators) to enhance receptor-mediated transport of biologically activeagents, including various bacterial products, cofactors and other activeagents disclosed herein, and analogs, variants, derivatives and mimeticsthereof. Generally, these ligands include hormones and growth factors,bacterial adhesins and toxins, lectins, metal ions and their carriers,vitamins, immunoglobulins, whole viruses and bacteria or selectedcomponents thereof. Exemplary ligands among these classes include, forexample, calcitonin, prolactin, epidermal growth factor, glucagon,growth hormone, estrogen, lutenizing hormone, platelet derived growthfactor, thyroid stimulating hormone, thyroid hormone, cholera toxin,diptheria toxin, E. coli heat labile toxin, Staphylococcal enterotoxinsA and B, ricin, saporin, modeccin, nigrin, sarcin, concanavalin A,transcobalantin, catecholamines, transferrin, folate, riboflavin,vitamin B1, low density lipoprotein, maternal IgO, polymeric IgA,adenovirus, vesicular stomatitis virus, Rous sarcoma virus, V. cholerae,Kiebsiella strains, Serratia strains, parainfluenza virus, respiratorysyncytial virus, Varicella zoster, and Enterobacter strains (see, forexample, Swann, Pa., Pharmaceutical Research 15:826-832, 1998,incorporated herein by reference).

In certain additional embodiments, membrane-permeable peptides (forexample, “arginine rich peptides”) are employed to facilitate deliveryof biologically active agents. While the mechanism of action of thesepeptides remains to be fully elucidated, they provide useful deliveryenhancing adjuncts for use within the compositions and methods herein.In one example, a basic peptide derived from human immunodeficiencyvirus (HIV)-1 Tat protein (for example, residues 48-60) has beenreported to translocate effectively through cell membranes andaccumulate in the nucleus, a characteristic which can be utilized forthe delivery of exogenous proteins and peptides into cells. The sequenceof Tat (GRKKRRQRRRPPQ, SEQ ID NO: 1) includes a highly basic andhydrophilic peptide, which contains 6 arginine and 2 lysine residues inits 13 amino acid residues. Various other arginine-rich peptides havebeen identified which have a translocation activity very similar toTat-(48-60). These include such peptides as the D-amino acid- andarginine-substituted Tat-(48-60), the RNA-binding peptides derived fromvirus proteins, such as HIV-1 Rev, and flock house virus coat proteins,and the DNA binding segments of leucine zipper proteins, such ascancer-related proteins c-Fos and c-Jun, and the yeast transcriptionfactor GCN4 (see, for example, Futaki et al., Journal BiologicalChemistry 276:5836-5840, 2000, incorporated herein by reference). Thesepeptides reportedly have several arginine residues marking their onlyidentified common structural characteristic, suggesting a commoninternalization mechanism ubiquitous to arginine-rich peptides, which isnot explained by typical endocytosis. Using (Arg)n (n=4-16) peptides,Futaki et al. teach optimization of arginine residues (n˜8) forefficient translocation. Recently, methods have been developed for thedelivery of exogenous proteins into living cells with the help ofarginine rich membrane-permeable carrier peptides such as HIV-1 Tat- andAntennapediasee, Futaki et al., supra, and references cited therein,incorporated herein by reference). By genetically or chemicallyhybridizing these carrier peptides with biologically active agents asdescribed herein, additional methods and compositions are thus providedto enhance delivery.

It will be understood by those skilled in the art that while thecompounds of the present disclosure will typically be employed asselective agonists or antagonists, there will be instances where acompound with a mixed steroid receptor profile is desired. For example,use of a PR agonists (for example, progestin) in female contraceptionoften leads to the undesired effects of increased water retention andacne. In this instance, a compound that is primarily a PR agonist, butalso displays some AR and MR modulating activity, can prove useful.Specifically, the mixed MR effects would be useful to control waterbalance in the body, while the AR effects would help to control any acneflare ups that occur.

Furthermore, it will be understood by those skilled in the art that thecompounds of the present disclosure, including pharmaceuticalcompositions and formulations containing these compounds, can be used ina wide variety of combination therapies to treat various conditions anddiseases as described herein. Thus, the compounds of the presentdisclosure can be used in combination with other active agents and othertherapies, including, without limitation, chemotherapeutic agents suchas cytostatic and cytotoxic agents, immunological modifiers such asinterferons, interleukins, growth hormones and other cytokines, hormonetherapies, surgery and radiation therapy.

A method of identifying a test agent that modulates LF blockade of theGR comprising: (a) obtaining cells that express the following: 1) GR; 2)an GR substrate, a GR reporter construct capable of measuring GRactivity (such as GR pathway activation), or both a GR substrate and aGR reporter construct; (b) subjecting the cells to a test agent; (c)measuring the amount of GR activity, wherein activity of the GR is usedto identify a test agent that modulates LF blockade of the GR. In oneexample, the ability of the agent to affect GR activity, but not toalter GR receptor number, identifies the agent as being of use.

The “glucocorticoid receptor” (GR) is a steroid hormone activatedtranscriptional factor known to regulate, either directly or indirectly,target genes involved in glucose homeostasis, bone turnover, celldifferentiation, lung maturation, and inflammation (Reichardt et al.,Adv. Pharmacol., 47:1-21, 2000). Mutations in GR are associated withCushing's syndrome, autoimmune diseases, and various cancers (Warner etal., Steroids, 61: 216-221, 1996). As such, GR is widely recognized as atherapeutically important target. GR ligands, including dexamethasone,prednisolone, and other related corticosteroid analogs, are commonlyused to treat diverse medical conditions such as asthma, allergicrhinitis, rheumatoid arthritis, and leukemia (Barnes et al., Am. J.Respir. Crit. Care Med., 157:S1-53, 1998). However, clinical use of oralcorticosteroids is limited by a number of side effects ranging fromincreased bone loss and growth retardation to suppression of thehypothalamic-pituitary-adrenal axis. Discovery of a GR agonist thatretains the beneficial anti-inflammatory activities without theundesired side effects is the subject of intense pharmaceutical efforts.

As noted above, GR belongs to the nuclear hormone receptor (NR)superfamily, which includes receptors for the mineralocorticoids (MR),estrogens (ER), progestins (PR), and androgens (AR), as well asreceptors for peroxisome proliferators (PARs), vitamin D (VDR), andthyroid hormones (TR). Phylogenetic analysis and sequence alignmentsshow that GR, MR, PR, and AR form a subfamily of oxosteroid receptorsthat are distinct from the ER subfamily (NRNC, 1999). These analysis areuseful for evaluating structure-function relationships between GR andits cognate ligands and cofactors.

Like most nuclear hormone receptors, GR is a modular protein that isorganized into three major domains: an N-terminal activation function-1domain (AF-1), a central DNA binding domain (DBD), and a C-terminalligand binding domain (LBD). In addition to its role in ligandrecognition, the LBD contains a ligand-dependent activation function(AF-2) that is tightly regulated by hormone binding.

Within the context of the full-length receptor, both the Ar-1 functionand the DNA binding activity of GR are dependent on hormone binding. Inthe absence of ligand, GR is retained in the cytoplasm by associationwith chaperone proteins such as hsp90 and p23, which bind to the LBD(Pratt et al., Endocr. Rev. 18:306-360, 1997). The chaperone activity ofthe hsp90 complex has been shown to be critical for hormone binding byGR (Bresnick et al., J. Biol. Chem., p. 4992-4997, 1989; Picard et al.,Nature, 348:166-168, 1990). Hormone binding initiates the release ofchaperone proteins from GR, allowing dimerization and translocation ofthe receptor into the nucleus. In the nucleus, GR binds to DNA promoterelements and can either activate or repress transcription depending onthe context of the target promoters. In addition, GR can also crosstalkwith other 110 transcriptional factors such as nuclear factor-κB (NF-κB)and activator protein-1 (AP-1) to repress their gene activationactivities (reviewed in McKay et al., Endocr. Rev., 20:435-459, 1999).This GR mediated repression has been postulated to be a molecular basisfor the anti-inflammatory and immunosuppressive activities ofglucocorticoids. Both the ligand-dependent activation and repression byGR require the intact function of the LBD.

The molecular mechanism of ligand-dependent regulation of nuclearhormone receptors has been illustrated by crystal structures of morethan a dozen NR LBDs that are either in the apo-state or bound toagonists or antagonists (Bourguet et al., Nature 375, 377-382 1995;Brzozowski et al., Nature 389, 753-758, 1997; Renaud et al., Nature 378,681-689 1995; Wagner et al., Nature 378 690-697 1995; Xu et al., Nature415, 813-817 1999). These analysis are also useful for evaluatingstructure-function relationships between GR and its cognate ligands andcofactors. The reported structures not only reveal that the LBDs foldinto a canonical three-layer helical sandwich that embeds a hydrophobicpocket for ligand binding, but also highlight the importance of theC-terminal (AF-2) helix in ligand dependent regulation. In the apo- orantagonist-bound receptor, the AF-2 helix is destabilized from its“active” conformation to allow the LBD to interact with co-repressorssuch as nuclear co-repressor (N-CoR) and silencing mediator for retinoidand thyroid hormone receptors (SMRT; Clen and Evans, Nature 377:454-457,1995; Horlein et al., Nature 377:397-404, 1995). Agonist binding inducesa conformational change of the AF-2 helix, stabilizing the receptor inan active conformation to facilitate its association with coactivatorproteins, such as steroid receptor coactivator-1 (SRC-1) andtranscriptional intermediary factor 2 (TIF2; Onate et al., Science270:1354-1357, 1995; Voegel et al., EMBO J. 17:507-519, 1996). Theseco-activators contain multiple LXXLL motifs, which interact with the NRLBD (Heery et al., Nature 387:733-736, 1997; Le Douarin et al., EMBO J.15:6701-6715, 1996). Various crystal structures of receptor/co-activatorpeptide complexes have revealed a general mode of coactivator binding toNRs. In these structures, the coactivator LXXLL motifs adopt a two-tum ahelix and both helical ends are stabilized by a “charge clamp” formed inpart by a conserved acidic residue from the AF-2 helix (Dan′-mont etal., Genes Dev. 12:3343-3356, 1998; Nolte et al., Nature 395:137-143,1998; Shiau et al., Cell 95:927-937, 1998).

Given its biological and pharmaceutical importance, there has beenenormous interest in elucidating the GR LBD structure. However, thesestructural efforts have been hampered by the inability to obtain apurified receptor that retains ligand binding activity. In a recentreport, die purification, crystallization, and structure determinationof the GR LBD in complex with dexamethasone and a co-activator motifderived from the cofactor TIF2 is described (Bledsoe et al., Cell110:93-105, 2002. Surprisingly, the structure reveals a novel dimerinterface unlike that observed for any other nuclear hormone receptor.Mutagenesis studies support the importance of this dimer interface in GRfunction. The crystal structure also reveals an unanticipated secondcharge clamp that is responsible for the specificity for the third TIF2LXXLL motif, and a distinct steroid binding pocket with features thatexplain ligand binding and selectivity. Since GR is highly homologous toMR, AR, and PR, the structure presented in this report serves as a modelfor understanding the roles of ligand binding, co-activator recruitment,and receptor dimerization in the signaling pathways mediated by thesesteroid receptors.

The glucocorticoid receptor is essential for survival and also formodulation of immune responses to infectious agents important inprotecting against lethal effects of bacteria, such as septic shock.Loss of activity of the glucocorticoid receptor during infection couldrender the host more susceptible to the lethal or toxic effects ofanthrax bacteria. Considering the mechanistic and therapeutic aspectsdisclosed herein, the findings herein indicate that simultaneous massivestimulation of cytokine release during anthrax infection, coupled withLF/LeTx repression of GR and other nuclear hormone receptors contributeto more severe consequences of infection including septic shock,increased stress and mortality, and exacerbated long-term sequelae dueto the removal of the anti-inflammatory effects of the glucocorticoidsreleased in response to infection. This scenario is consistent with thewell-described increased mortality from septic shock in animals exposedto both glucocorticoid receptor antagonists and infectious agents orpro-inflammatory bacterial products. GR repression by LF/PA also likelycontributes to the chronic fatigue syndrome-like symptoms, cognitive andinflammatory symptoms now being reported in relation to anthraxexposure, since blunted glucocorticoid responses have been associatedwith many inflammatory diseases, cognitive symptoms and fatigue states.Thus, in one embodiment, an agent that alters GR activity can be used toalter an immune response to an infectious agent.

Simultaneous loss of activity or enhancement of activity of othernuclear hormone receptors, including PR, and resulting imbalance inratios of relative activity of nuclear hormone receptors likelyamplifies these immune enhancing effects. Identification of nuclearhormone receptor co-factor interactions as a mechanism of toxicity ofanthrax lethal factor and other bacterial products (such as bacterialtoxins and antigens such as superantigens (SAgs) will therefore providenew tools and methods for treatment and prevention of the toxic effectsof anthrax and other pathogenic infections. In more detailed aspects,these tools will be effective to minimize adverse side effects ofinfection, including toxicities, inflammatory symptoms, or relatedcomplications, including autoimmune diseases exacerbated by nuclearhormone receptor repression (for example, lupus, rheumatoid arthritis(RA), diabetes mellitus, multiple sclerosis, regional enteritis, thyroidcancer, and other diseases and conditions).

In addition, the methods and compositions disclosed herein provide toolsfor identification, removal and/or avoidance of host and or vaccinefactors predisposing an individual to increased risk of adverse sequelaeassociated with pathogenic infection, inflammatory disorders andautoimmune disease. In the case of bacterial infection, products thatrepress nuclear hormone receptors are likely to account for idiopathicchronic fatigue syndromes, inflammatory arthritis and autoimmunediseases, and potentially for lethal and septic shock effects of certainbacterial strains. These products may also account for some ubiquitousidiopathic chronic inflammatory or fatigue symptoms unrelated toinfectious exposures.

The disclosure concerning molecular interactions of the anthrax lethalfactor with GR identify a novel mechanism by which the lethal toxin ofBacillus anthracis (anthrax LeTx), interferes with a number of nuclearhormone receptors essential for life and healthy functioning of cells.These findings have immediate important public health implications notonly for anthrax infection and biodefense, but are also potentiallyrelevant to explain toxicities related to a wide range of bacterialproducts and for the development of potential therapeutic interventionsto prevent and treat toxic sequelae of infection with such pathogens.

In other embodiments, the selective and specific effects of LeTx andother bacterial products on range of nuclear hormone receptors makethese products useful tools for elucidating the molecular mechanisms ofinteractions between bacterial products and nuclear hormone receptorsand their co-factors.

Agents that affect the activity of a nuclear hormone receptor, asdisclosed herein, are useful to influence basic, life sustaining systemsof the body, including carbohydrate, protein and lipid metabolism,electrolyte and water balance, and the functions of the cardiovascular,kidney, central nervous, immune, skeletal muscle and other organ andtissue systems. In this regard, GR and MR modulators (agonists andantagonists) have proved useful in the treatment of inflammation, tissuerejection, auto-immunity, hypertension, various malignancies, such asluekerias, lymphomas and breast and prostate cancers, Cushing'ssyndrome, glaucoma, obesity, rheumatoid arthritis, acute adrenalinsufficiency, congenital adrenal hyperplasia, osteoarthritis, rheumaticfever, systemic lupus erythematosus, polymyositis, polyarteritis nodosa,granulomatous polyarteritis, allergic diseases such as urticaria, drugreactions and hay fever, asthma, a variety of skin diseases,inflammatory bowel disease, hepatitis and cirrhosis. Accordingly, insome examples, GR and MR modulatory compounds are useful as immunostimulants and repressors, wound healing and/or tissue repair agents,catabolic/antianabolic activators, and as antibacterial or anti-viralagents (such as for treatment or prevention of symptoms related toanthrax, herpes simplex viral infection and related symptoms).

The bacterial products that modulate nuclear hormone receptor activity(including naturally occurring, recombinant, and synthetic peptides andproteins, and peptide and protein analogs and mimetics of nativebacterial products) can be used for screening (for example, in kitsand/or screening assay methods) to identify additional compounds,including other peptides, proteins, analogs and mimetics, that willfunction within the methods and compositions disclosed herein, includingas nuclear hormone receptor agonists and antagonists. Several methods ofautomating assays have been developed in recent years so as to permitscreening of tens of thousands of compounds in a short period (see, forexample, Fodor et al., Science 251:767-773, 1991, and U.S. Pat. Nos.5,677,195; 5,885,837; 5,902,723; 6,027,880; 6,040,193; and 6,124,102,issued to Fodor et al., each incorporated herein by reference). Largecombinatorial libraries of compounds can be constructed by encodedsynthetic libraries (ESL) described in, for example, WO 95/12608, WO93/06121, WO 94/08051, WO 95/35503, and WO 95/30642 (each incorporatedby reference). Peptide libraries can also be generated by phage displaymethods (see, for example, Devlin, WO 91/18980, incorporated herein byreference). Many other publications describing chemical diversitylibraries and screening methods are also considered reflective of thestate of the art pertaining to these aspects and are generallyincorporated herein.

One method of screening for agents that affect the activity of nuclearhormone receptors (such as to screen for small molecule drugs, LFanalogs, and peptide mimetics that reduce or block LF or LeTx repressionof GR or PR) utilizes eukaryotic or prokaryotic host cells which arestably transformed with recombinant DNA molecules expressing an activebacterial peptide or protein, for example, LF or LeTx. Such cells,either in viable or fixed form, can be used for standard assays, forexample, ligand/receptor binding assays (see, for example, Parce et al.,Science 246:243-247, 1989; and Owicki et al., Proc. Natl. Acad. Sci. USA87:4007-4011, 1990, each incorporated herein by reference). Competitiveassays are particularly useful, for example assays where the cells arecontacted and incubated with a labeled receptor, receptor ligand, DNAbinding target of the receptor, receptor cofactor, or antibody havingbinding affinity to the bacterial product or to an indirect bindingpartner that in turn binds the bacterial product. In conjunction withthese assays, a test compound may be added to detect interruption ofdirect or indirect binding interactions. Bound and free labeled bindingcomponents are typically separated to assess the degree of specificbinding and/or binding enhancement or inhibition. Any one of numeroustechniques can be used to separate bound from free agents to assess thedegree of binding (such as between a bacterial product and a cofactor ofa nuclear hormone receptor, between a cofactor and its cognate receptorin the presence or absence of a selected bacterial toxin, etc.) Thisseparation step can involve a conventional procedure such as adhesion tofilters followed by washing, adhesion to plastic followed by washing, orcentrifugation of the cell membranes.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto a target molecule, such as a labeled receptor, receptor ligand, DNAbinding target of the receptor, receptor cofactor, or antibody havingbinding affinity to the bacterial product or to an indirect bindingpartner that in turn binds the bacterial product. Representativescreening methods for use within these embodiments are provided, forexample, in Geysen, European Patent Application 84/03564, published onSep. 13, 1984 (incorporated herein by reference). First, large numbersof different test compounds, such as small peptides, are synthesized ona solid substrate, for example, plastic pins or some other appropriatesurface, (see, for example, Fodor et al., Science 251:767-773, 1991, andU.S. Pat. Nos. 5,677,195; 5,885,837; 5,902,723; 6,027,880; 6,040,193;and 6,124,102, issued to Fodor et al., each incorporated herein byreference). Then all of the pins are reacted with a solubilized peptideagent, and washed. The next step involves detecting bound peptide.

Rational drug design may also be based upon structural studies of themolecular shapes of biologically active peptides and proteins determinedto operate within the methods disclosed herein. Various methods areavailable and well known in the art for characterizing, mapping,translating, and reproducing structural features of peptides andproteins to guide the production and selection of new peptide mimetics,including for example x-ray crystallography and 2 dimensional NMRtechniques. These and other methods, for example, will allow reasonedprediction of which amino acid residues present in a selected peptide orprotein form molecular contact regions necessary for specificity andactivity (see, for example, Blundell and Johnson, ProteinCrystallography, Academic Press, N.Y., 1976, incorporated herein byreference).

Operable analogs and mimetics of bacterial products and of otherbiologically active agents disclosed herein retain partial, complete orenhanced activity compared to a native peptides, protein or unmodifiedcompound. For example analogs or mimetics of LF or LeTx will exhibitpartial or complete activity for nuclear hormone receptor repression. Inthis regard, operable analogs and mimetics for use will often retain atleast 50%, often 75%, and up to 95-100% or greater levels of one or moreselected activities as compared to the same activity observed for aselected native peptide or protein or unmodified compound. Thesebiological properties of altered peptides or non-peptide mimetics can bedetermined according to any suitable assay disclosed or incorporatedherein, for example by determining the ability of a LF peptide ormimetic to repress GR activation. Where bacterial products arecontemplated for use as therapeutics, they will typically be engineeredfor reduced toxicity.

In accordance with the description herein, the compounds disclosedherein are useful in vitro as unique tools for analyzing the nature andfunction of interactions between bacterial products and members ofnuclear hormone receptor activation and repression pathways. Thesecompounds will therefore also serve as leads in various programs fordesigning additional peptide and non-peptide (for example, smallmolecule drug) agents for regulating activation and repression ofnuclear hormone receptor activity, including in clinical contexts totreat or prevent disease and other conditions associated with aberrantfunctioning of one or more nuclear hormone receptors.

Those skilled in the art will readily appreciate that a wide range ofadditional screening assays can be employed to identify moleculescapable of modulating one or more activities (such as ligand binding,DNA binding, expression of nuclear hormone receptor regulated endogenousgenes or reporter constructs) of, for example, a bacterial product,nuclear hormone receptor, receptor ligand, DNA binding target of thereceptor, or receptor cofactor. Such assays can involve theidentification of compounds that interact with these and other compoundsof interest, either physically (for example, by binding) or genetically,and can thus rely on any of a number of standard methods to detectphysical or genetic interactions between multiple subject compounds.Such assays can also involve the identification of compounds that affectexpression, activity or other properties, such as phosphorylation ornuclear localization, of the subject compound(s) or ability to bind yetadditional binding partners, including labeled binding partners such asantibodies. Such assays can be cell-free or cell-based, and the lattertype of assays can be performed in any type of cell, such as a cell thatnaturally or artificially incorporates or expresses one or more productsof interest, for example one or more bacterial product(s), nuclearhormone receptor(s), receptor ligand(s), DNA binding target(s) of thereceptor, receptor cofactor(s), etc.

Compounds that are involved in activation or repression of nuclearhormone receptor pathways can be identified and/or isolated based on anability to specifically bind to a screening compound of interest, forexample a bacterial product, nuclear hormone receptor, receptor ligand,DNA binding target of the receptor, or receptor cofactor. Likewise,screening methods for use can be based on binding to a fragment orconjugate of one of these subject compounds, or by binding to anantibody that likewise recognizes the subject compound. In numerousembodiments, the subject compound will be attached to a solid support.In one embodiment, affinity columns are made using the subject compoundand physically-interacting molecules are identified. It will be apparentto one of skill that chromatographic techniques can be performed at anyscale and using equipment from many different manufacturers. Inaddition, molecules that interact with subject compounds in vivo can beidentified by co-immunoprecipitation or other methods, for example,immunoprecipitating subject bacterial proteins or cofactors usinganti-antibodies to pull the subject compound(s) from a cell or cellextract, and identifying candidate compounds that bind the subjectcompounds that are precipitated along with the subject protein. Suchmethods are well known to those of skill in the art.

Two-hybrid screens can also be used to identify polypeptides thatinteract in vivo with a subject compound (see, for example, Fields etal., Nature 340:245-246, 1989). Such screens comprise two discrete,modular domains of a transcription factor protein, for example, a DNAbinding domain and a transcriptional activation domain, which areproduced in a cell as two separate polypeptides, each of which alsocomprises one of two potentially binding polypeptides. If the twopotentially binding polypeptides (for example, a bacterial toxin and acofactor of a nuclear hormone receptor) in fact interact in vivo, thenthe DNA binding and the transcriptional activating domain of thetranscription factor are united, thereby producing expression of atarget gene in the cell. The target gene typically encodes an easilydetectable gene product, for example, β-galactosidase, GFP, orluciferase, which can be detected using standard methods. In oneexemplary embodiment, a LF polypeptide is fused to one of the twodomains of the transcription factor, and a known or potential nuclearhormone receptor cofactor polypeptide (for example, encoded by a cDNAlibrary) is fused to the other domain. Such methods are well known tothose of skill in the art.

In other embodiments, transcription levels can be measured to assess dieeffects of a test compound on nuclear hormone receptor pathway activity.In various examples, a host cell containing a nuclear hormone receptorof interest is transformed to express a “reporter construct” that yieldsa detectable signal for receptor pathway activity. Alternatively or incombination with this protocol, the cell may be contacted with, orgenetically engineered to express, one or more of the following: anative or modified (such as a truncated mutant, chimeric, or tagged)receptor, receptor ligand, DNA binding target of the receptor, or areceptor cofactor, which are “substrates.” The cell is then exposed to atest compound for a sufficient time to effect any binding or otherinteractions between the test compound and subject compounds, and thenthe interactions are detected (such as by immunoprecipitation, detectionof levels of gene expression, etc.) Levels of transcription may bemeasured using any method known to those of skill in the art to besuitable. For example, mRNA expression of a protein of interest may bedetected using Northern blots or by detecting their polypeptide productsusing immunoassays. Many polynucleotides typically expressed followingnuclear hormone receptor activation will thus be detectable. (see, forexample, Lenardo, et al., Cell 58:227, 1989; Grilli, et al., Int. Rev.Cytol. 143:1, 1993; Baeuerle, et al., Ann. Rev. Immunol. 12:141, 1994.Such assays can use natural targets, for example targets of NF-κB or canuse reporter genes, such as chloramphenicol acetyltransferase,luciferase, β-galactosidase, GFP, and alkaline phosphatase, operablylinked to a promoter containing a binding site for a compound ofinterest (for example, a ligand of a nuclear hormone receptor).Furthermore, a protein of interest can be used as an indirect reportervia attachment to a second reporter such as green fluorescent protein(see, for example, Mistili & Spector, Nature Biotechnology 15:961-964,1997.

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks one or more of the compound(s)interest (such as a cell that does not have an expression constructdirecting expression of a nuclear hormone receptor cofactor introducedinto the test cell). A substantially identical cell may be derived fromthe same cells from which the recombinant cell was prepared but whichhad not been modified by introduction of heterologous DNA. Anydifference in the amount of transcription indicates that the testcompound has in some manner altered the activity of the protein ofinterest.

Compounds tested as modulators of nuclear hormone receptor activity caninclude any small chemical compound, or a biochemical compound such as aprotein, peptide, protein, sugar, nucleic acid or lipid. Other testcompounds will comprise a recombinantly or genetically modified nuclearhormone receptor, receptor ligand, DNA binding target of a receptor,receptor cofactor, or the like. Typically, test compounds will be smallchemical molecules and peptides. Essentially any chemical compound canbe used as a potential modulator or binding compound in the assaysdisclosed herein, although most often compounds that can be dissolved inaqueous or organic (especially DMSO-based) solutions are used. Theassays are designed to screen large chemical libraries by automating theassay steps and providing compounds from any convenient source toassays, which are typically run in parallel (for example, in microtiterformats on microtiter plates in robotic assays). It will be appreciatedthat there are many suppliers of chemical compounds, including Sigma(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis,Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and thelike.

In one embodiment, high throughput screening methods involve providing acombinatorial chemical or peptide library containing a large number ofpotential therapeutic compounds (potential modulator or bindingcompounds). Such “combinatorial chemical libraries” are then screened inone or more assays, as described herein, to identify those librarymembers (particular chemical species or subclasses) that display adesired characteristic activity. The compounds thus identified can serveas conventional “lead compounds” or can themselves be used as potentialor actual therapeutics.

A “combinatorial chemical library” is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(for example, the number of amino acids in a polypeptide compound).Millions of chemical compounds can be synthesized through suchcombinatorial mixing of chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, forexample, U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res.,37:487-493, 1991 and Houghton et al., Nature, 354:84-88, 1991). Otherchemistries for generating chemical diversity libraries can also beused. Such chemistries include, but are not limited to: peptoids (forexample, see PCT Publication No. WO 91/19735), encoded peptides (forexample, PCT Publication No. WO 93/20242), random bio-oligomers (forexample, see PCT Publication No. WO 92/00091), benzodiazepines (forexample, see U.S. Pat. No. 5,288,514), diversomers such as hydantoins,benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA90, 6909-6913, 1993), vinylogous polypeptides (Hagihara et al., J. Amer.Chem. Soc., 114:65-68, 1992), nonpeptidal peptidomimetics with glucosescaffolding (Hirschmann et al., J. Amer. Chem. Soc., 114:9217-9218,1992), analogous organic syntheses of small compound libraries (Chen etal., J. Amer. Chem. Soc. 116:2661, 1994), oligocarbamates (Cho et al.,Science, 61:1303, 1993), and/or peptidyl phosphonates (Campbell et al.,J. Org. Chem., 59:658, 1994), nucleic acid libraries and peptide nucleicacid libraries (for example, see U.S. Pat. No. 5,539,083), antibodylibraries (see, for example, Vaughn et al., Nature Biotechnology,14:309-314, 1996 and PCT/US96/10287), carbohydrate libraries (forexample, see Liang et al., Science, 274:1520-1522, 1996 and U.S. Pat.No. 5,593,853), small organic molecule libraries for example,benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids,U.S. Pat. No. 5,569,588; thiazolidinones and metatliazanones, U.S. Pat.No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S.Pat. No. 5,288,514, and the like.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, for example, 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, for example, ComGenex, Princeton, N.J.,Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa., MartekBiosciences, Columbia, Md., etc.)

In the high throughput assays disclosed herein, it is possible to screenup to several thousand different subject compounds in a single day. Inparticular, each wen of a microtiter plate can be used to run a separateassay against a selected potential nuclear hormone receptor modulator,or, if concentration or incubation time effects are to be observed,every 5-10 wells can test a single modulator. Thus, a single standardmicrotiter plate can assay about 100 (for example, 96) modulators. If1536 well plates are used, then a single plate can easily assay fromabout 100 to about 1500 different compounds. It is possible to assayseveral different plates per day; assay screens for up to about6,000-20,000 different compounds is possible using the integratedsystems disclosed herein. More recently, microfluidic approaches toreagent manipulation have been developed.

The molecule of interest can be bound to a solid state component,directly or indirectly, via covalent or non covalent linkage, such asvia a tag. The tag can be any of a variety of components.

In general, a molecule which binds the tag (a tag binder) is fixed to asolid support, and the tagged molecule of interest is attached to thesolid support by interaction of the tag and the tag binder. A number oftags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMAInmaunochemicals 1998 catalogue SIGMA, St. Louis Mo.). Similarly, anyhaptenic or antigenic compound can be used in combination with anappropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. Synthetic polymers,such as polyurethanes, polyesters, polycarbonates, polyureas,polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes,polyimides, and polyacetates can also form an appropriate tag or tagbinder. Many other tag/tag binder pairs are also useful in assay systemsdescribed herein, as would be apparent to one of skill upon review ofthis disclosure. Common linkers such as peptides, polyethers, and thelike can also serve as tags, and include polypeptide sequences, such aspoly-gly sequences of between about 5 and 200 amino acids. Such flexiblelinkers are known to persons of skill in the art. For example,poly(ethelyne glycol) linkers are available from Shearwater Polymers,Inc. Huntsville, Ala. These linkers optionally have amide linkages,sulfhydryl linkages, or heterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, for example,Merrifield, J. Am. Chem. Soc., 85:2149-2154, 1963, (describing solidphase synthesis of, for example, peptides); Geysen et al., J. Immun.Meth., 102:259-274, 1987 (describing synthesis of solid phase componentson pins); Frank et al., Tetrahedron 44:6031-6040, 1988 (describingsynthesis of various peptide sequences on cellulose disks); Fodor etal., Science, 251:767-777, 1991; Sheldon, et al., Clinical Chemistry,39:718-719, 1993; and Kozal et al., Nature Medicine, 2:753-759, 1996(all describing arrays of biopolymers fixed to solid substrates).Nonchemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Yet another assay for compounds that modulate nuclear hormone receptoractivity involves computer assisted drug design, in which a computersystem is used to generate a three-dimensional structure of, forexample, a bacterial product, nuclear hormone receptor, receptor ligand,or receptor cofactor based, for example on the structural informationencoded by its amino acid sequence. The input amino acid sequenceinteracts directly and actively with a preestablished algorithm in acomputer program to yield secondary, tertiary, and quaternary structuralmodels of the protein. The models of the protein structure are thenexamined to identify regions of the structure that have the ability tobind. These regions are then used to identify compounds that bind to theprotein. The three-dimensional structural model of the protein isgenerated by entering protein amino acid sequences of at least 10 aminoacid residues or corresponding nucleic acid sequences encoding a subjectpolypeptide into the computer system. The nucleotide sequence encodingthe polypeptide may, for example, comprise a sequence encoding a portionof a bacterial product or nuclear hormone receptor cofactor, or aconservatively modified version thereof. At least 10 residues of theamino acid sequence (or a nucleotide sequence encoding 10 amino acids)are entered into the computer system from computer keyboards, computerreadable substrates that include, but are not limited to, electronicstorage media (for example, magnetic diskettes, tapes, cartridges, andchips), optical media (for example, CD ROM), information distributed byinternet sites, and by RAM. The three-dimensional structural model ofthe protein is then generated by the interaction of the amino acidsequence and the computer system, using software known to those of skillin the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, for example, cytoplasmic, surface, ornuclear. These variables along with the energy terms of the secondarystructure are used to form the model of the tertiary structure. Inmodeling the tertiary structure, the computer program matcheshydrophobic faces of secondary structure with like, and hydrophilicfaces of secondary structure with like.

Once the structure has been generated, potential modulator bindingregions are identified by the computer system. Three-dimensionalstructures for potential modulators are generated by entering amino acidor nucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential modulator isthen compared to that of the subject protein to identify compoundslikely to bind to the protein. Binding affinity between the protein andcompound is determined using energy terms to determine which compoundshave an enhanced probability of binding to the protein.

In numerous embodiments, a compound, for example, nucleic acid,polypeptide, or other molecule is administered to a patient, in vivo orex vivo, to effect a change in nuclear hormone receptor activity orexpression in the patient. Such compounds can include nucleic acidsencoding any of the compounds of interest identified herein or selectedaccording to the screening methods disclosed herein (as well asrecombinantly modified derivatives, fragments, variants, or fusionsthereof), operably linked to a promoter. Suitable nucleic acids alsoinclude inhibitory sequences such as antisense or ribozyme sequences,which can be delivered in, for example, an expression vector operablylinked to a promoter, or can be delivered directly. Also, any nucleicacid that encodes a polypeptide that modulates the expression of anuclear hormone receptor can be used. In general, nucleic acids can bedelivered to cells using any of a large number of vectors or methods,for example, retroviral, adenoviral, or adeno-associated virus vectors,liposomal formulations, naked DNA injection, and others. All of thesemethods are well known to those of skill in the art.

The therapeutic compounds, for example native or modified bacterialproducts, nuclear hormone receptors, receptor cofactors, and antibodieshaving binding affinity to a bacterial product or cofactor, aregenerally provided for direct administration to subjects in asubstantially purified form. The term “substantially purified” as usedherein, is intended to refer to a peptide, protein, nucleic acid orother compound that is isolated in whole or in part from naturallyassociated proteins and other contaminants, wherein the peptide,protein, nucleic acid or other active compound is purified to ameasurable degree relative to its naturally-occurring state, forexample, relative to its purity within a cell extract.

In certain embodiments, the term “substantially purified” refers to apeptide, protein, or polynucleotide composition that has been isolatedfrom a cell, cell culture medium, or other crude preparation andsubjected to fractionation to remove various components of the initialpreparation, such as proteins, cellular debris, and other components. Ofcourse, such purified preparations may include materials in covalentassociation with the active agent, such as glycoside residues ormaterials admixed or conjugated with the active agent, which may bedesired to yield a modified derivative or analog of the active agent orproduce a combinatorial therapeutic formulation, conjugate, fusionprotein or the like. The term purified thus includes such desiredproducts as peptide and protein analogs or mimetics or otherbiologically active compounds wherein additional compounds or moietiessuch as polyethylene glycol, biotin or other moieties are bound to theactive agent in order to allow for the attachment of other compoundsand/or provide for formulations useful in therapeutic treatment ordiagnostic procedures.

As applied to polynucleotides, the term substantially purified denotesthat the polynucleotide is free of substances normally accompanying it,but may include additional sequence at the 5′ and/or 3′ end of thecoding sequence which might result, for example, from reversetranscription of the noncoding portions of a message when the DNA isderived from a cDNA library, or might include the reverse transcript forthe signal sequence as well as the mature protein encoding sequence.

When referring to peptides, proteins and peptide analogs (includingpeptide fusions with other peptides and/or proteins) of use, the termsubstantially purified typically means a composition which is partiallyto completely free of other cellular components with which the peptides,proteins or analogs are associated in a non-purified, for example,native state or environment. Purified peptides and proteins aregenerally in a homogeneous or nearly homogenous state although it can beeither in a dry state or in an aqueous solution. Purity and homogeneityare typically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography.

Generally, substantially purified peptides, proteins and other activecompounds for use comprise more than 80% of all macromolecular speciespresent in a preparation prior to admixture or formulation of thepeptide, protein or other active agent with a pharmaceutical carrier,excipient, buffer, absorption enhancing agent, stabilizer, preservative,adjuvant or other co-ingredient in a complete pharmaceutical formulationfor therapeutic administration. More typically, the peptide or otheractive agent is purified to represent greater than 90%, often greaterthan 95% of all macromolecular species present in a purified preparationprior to admixture with other formulation ingredients. In other cases,the purified preparation of active agent may be essentially homogeneous,wherein other macromolecular species are not detectable by conventionaltechniques.

Therapeutic and prophylactic formulations can include a biologicallyactive subject compound as described above typically combined togetherwith one or more pharmaceutically acceptable carriers and, optionally,other therapeutic ingredients. The carrier(s) must be “pharmaceuticallyacceptable” in the sense of being compatible with the other ingredientsof the formulation and not eliciting an unacceptable deleterious effectin the subject. Such carriers are described herein above or areotherwise well known to those skilled in the art of pharmacology.Desirably, the formulation should not include substances such as enzymesor oxidizing agents with which the biologically active agent to beadministered is known to be incompatible. The formulations may beprepared by any of the methods well known in the art of pharmacy.

Within the compositions and methods disclosed herein, the active subjectcompound (including peptides, proteins, analogs and mimetics, and otherbiologically active agents disclosed herein) may be administered tosubjects by a variety of mucosal administration modes, including byoral, rectal, intranasal, intrapulmonary, or transdermal delivery, or bytopical delivery to other surfaces. Optionally, the active agentsdisclosed herein can be administered by non-mucosal routes, including byintramuscular, subcutaneous, intravenous, intra-atrial, intra-articular,intraperitoneal, or parenteral routes. In other alternative embodiments,the biologically active agent(s) can be administered ex vivo by directexposure to cells, tissues or organs originating from a mammaliansubject, for example as a component of an ex vivo tissue or organtreatment formulation that contains the biologically active agent in asuitable, liquid or solid carrier.

To formulate pharmaceutical compositions, the biologically active agentcan be combined with various pharmaceutically acceptable additives, aswell as a base or carrier for dispersion of the active agent(s). Desiredadditives include, but are not limited to, pH control agents, such asarginine, sodium hydroxide, glycine, hydrochloric acid, citric acid,etc. In addition, local anesthetics (for example, benzyl alcohol),isotonizing agents (for example, sodium chloride, mannitol sorbitol),adsorption inhibitors (for example, Tween 80), solubility enhancingagents (for example, cyclodextrins and derivatives thereof), stabilizers(for example, serum albumin), and reducing agents (for example,glutathione) can be included. When the composition for delivery is aliquid, the tonicity of the formulation, as measured with reference tothe tonicity of 0.9% (w/v) physiological saline solution taken as unity,is typically adjusted to a value at which no substantial, irreversibletissue damage will be induced in the nasal mucosa at the site ofadministration. Generally, the tonicity of the solution is adjusted to avalue of about ⅓ to 3, more typically ½ to 2, and most often ¾ to 1.7.

The biologically active agent can be dispersed in a base or vehicle,which may comprise a hydrophilic compound having a capacity to dispersethe active agent and any desired additives. The base can be selectedfrom a wide range of suitable carriers, including but not limited to,copolymers of polycarboxylic acids or salts thereof, carboxylicanhydrides (for example, maleic anhydride) with other monomers (forexample, methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinylpolymers such as polyvinyl acetate, polyvinyl alcohol,polyvinylpyrrolidone, cellulose derivatives such ashydroxymethylcellulose, hydroxypropylcellulose, etc., and naturalpolymers such as chitosan, collagen, sodium alginate, gelatin,hyaluronic acid, and nontoxic metal salts thereof. Often, abiodegradable polymer is selected as a base or carrier, for example,polylactic acid, poly(lactic acid-glycolic acid) copolymer,polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)copolymer and mixtures thereof. Alternatively or additionally, syntheticfatty acid esters such as polyglycerin fatty acid esters, sucrose fattyacid esters, etc. can be employed as carriers. Hydrophilic polymers andother carriers can be used alone or in combination, and enhancedstructural integrity can be imparted to the carrier by partialcrystallization, ionic bonding, crosslinking and the like. The carriercan be provided in a variety of forms, including, fluid or viscoussolutions, gels, pastes, powders, microspheres and films for directapplication to the nasal mucosa. The use of a selected carrier in thiscontext may result in promotion of absorption of the biologically activeagent.

The biologically active agent can be combined with the base or carrieraccording to a variety of methods, and release of the active agent maybe by diffusion, disintegration of the carrier, or associatedformulation of water channels. In some circumstances, the active agentis dispersed in microcapsules (microspheres) or nanocapsules(nanospheres) prepared from a suitable polymer, for example, isobutyl2-cyanoacrylate (see, for example, Michael et al., J. PharmacyPharmacol. 43: 1-5, 1991), and dispersed in a biocompatible dispersingmedium, which yields sustained delivery and biological activity over aprotracted time.

The compositions can alternatively contain as pharmaceuticallyacceptable carriers substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. For solidcompositions, conventional nontoxic pharmaceutically acceptable carrierscan be used which include, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like.

Therapeutic compositions for administering the biologically activeagent(s) can also be formulated as a solution, microemulsion, or otherordered structure suitable for high concentration of active ingredients.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. Proper fluidity for solutions can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofa desired particle size in the case of dispersible formulations, and bythe use of surfactants. In many cases, it will be desirable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe biologically active agent can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin.

In certain embodiments, the biologically active agent is administered ina time release formulation, for example in a composition which includesa slow release polymer. The active agent can be prepared with carriersthat will protect against rapid release, for example a controlledrelease vehicle such as a polymer, microencapsulated delivery system orbioadhesive gel. Prolonged delivery of the active agent, in variouscompositions disclosed herein, can be brought about by including in thecomposition agents that delay absorption, for example, aluminummonosterate hydrogels and gelatin. When controlled release formulationsof the biologically active agent is desired, controlled release binderssuitable for use include any biocompatible controlled-release materialwhich is inert to the active agent and which is capable of incorporatingthe biologically active agent. Numerous such materials are known in theart. Useful controlled-release binders are materials that aremetabolized slowly under physiological conditions following theirdelivery. Appropriate binders include but are not limited tobiocompatible polymers and copolymers previously used in the art insustained release formulations. Such biocompatible compounds arenon-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody.

Exemplary polymeric materials for use in this context include, but arenot limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolysable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids (PGA) and polylactic acids (PLA),poly(DL-lactic acid-co-glycolic acid)(DL PLGA), poly(D-lacticacid-coglycolic acid)(D PLGA) and poly(L-lactic acid-co-glycolic acid)(LPLGA). Other useful biodegradable or bioerodable polymers include butare not limited to such polymers as poly(epsilon-caprolactone),poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly (ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters,polycarbonate, polymaleamides, polysaccharides and copolymers thereof.Many methods for preparing such formulations are generally known tothose skilled in the art (see, for example, Sustained and ControlledRelease Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,New York, 1978, incorporated herein by reference). Other usefulformulations include controlled-release compositions such as are knownin the art for the administration of leuprolide (trade name:Lupron.RTM.), for example, microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893, each incorporated herein by reference), lactic acid-glycolicacid copolymers useful in making microcapsules and other formulations(U.S. Pat. Nos. 4,677,191 and 4,728,721, each incorporated herein byreference), and sustained-release compositions for water-solublepeptides (U.S. Pat. No. 4,675,189, incorporated herein by reference).

The pharmaceutical formulations typically must be sterile and stableunder all conditions of manufacture, storage and use. Sterile solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The prevention of the action ofmicroorganisms can be accomplished by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

In more detailed aspects, the biologically active agent is stabilized toextend its effective half-life following delivery to the subject,particularly for extending metabolic persistence in an active statewithin the physiological environment (for example, at a mucosal surface,in the bloodstream, or within a connective tissue compartment orfluid-filled body cavity). For this purpose, the biologically activeagent may be modified by chemical means, for example, chemicalconjugation, N-terminal capping, PEGylation, or recombinant means, forexample, site-directed mutagenesis or construction of fusion proteins,or formulated with various stabilizing agents or carriers. Thusstabilized, the active agent administered as above retains biologicalactivity for an extended period (for example, 2-3, up to 5-10 foldgreater stability) under physiological conditions compared to itsnon-stabilized form.

In accordance with the various treatment methods disclosed herein, thebiologically active agent is delivered to a mammalian subject in amanner consistent with conventional methodologies associated withmanagement of the disorder for which treatment or prevention is sought.In accordance with the disclosure herein, a prophylactically ortherapeutically effective amount of the biologically active agent isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent, inhibit, and/or ameliorate a selecteddisease or condition or one or more symptom(s) thereof.

The term “subject” as used herein means any mammalian patient to whichthe compositions can be administered. Typical subjects intended fortreatment with the compositions and methods disclosed herein includehumans, as well as non-human primates and other animals. To identifysubject patients for prophylaxis or treatment according to the methodsdisclosed herein, accepted screening methods are employed to determinerisk factors associated with a targeted or suspected disease ofcondition as discussed above, or to determine the status of an existingdisease or condition in a subject. These screening methods include, forexample, conventional work-ups to determine familial, sexual, drug-useand other such risk factors that may be associated with the targeted orsuspected disease or condition, as well as diagnostic methods such asvarious ELISA immunoassay methods, which are available and well known inthe art to detect and/or characterize disease-associated markers. Theseand other routine methods allow the clinician to select patients in needof therapy using the methods and formulations disclosed herein. Inaccordance with these methods and principles, biologically active agentsmay be administered according to the teachings herein as an independentprophylaxis or treatment program, or as a follow-up, adjunct orcoordinate treatment regimen to other treatments, including surgery,vaccination, immunotherapy, hormone treatment, cell, tissue, or organtransplants, and the like.

For prophylactic and treatment purposes, the biologically activeagent(s) disclosed herein may be administered to the subject in a singlebolus delivery, via continuous delivery (for example, continuoustransdermal, mucosal, or intravenous delivery) over an extended timeperiod, or in a repeated administration protocol (for example, by anhourly, daily or weekly, repeated administration protocol). In thiscontext, a therapeutically effective dosage of the biologically activeagent(s) may include repeated doses within a prolonged prophylaxis ortreatment regimen, that will yield clinically significant results toalleviate one or more symptoms or detectable conditions associated witha targeted disease or condition as set forth above. Determination ofeffective dosages in this context is typically based on animal modelstudies followed up by human clinical trials and is guided bydetermining effective dosages and administration protocols thatsignificantly reduce the occurrence or severity of targeted diseasesymptoms or conditions in the subject. Suitable models in this regardinclude, for example, murine, rat, porcine, feline, non-human primate,and other accepted animal model subjects known in the art.Alternatively, effective dosages can be determined using in vitro models(for example, immunologic and histopathologic assays). Using suchmodels, only ordinary calculations and adjustments are typicallyrequired to determine an appropriate concentration and dose toadminister a therapeutically effective amount of the biologically activeagent(s) (for example, amounts that are intranasally effective,transdermally effective, intravenously effective, or intramuscularlyeffective to elicit a desired response). In alternative embodiments, an“effective amount” or “effective dose” of the biologically activeagent(s) may simply inhibit or enhance one or more selected biologicalactivity(ies) correlated with a disease or condition, as set forthabove, for either therapeutic or diagnostic purposes.

The actual dosage of biologically active agents will of course varyaccording to factors such as the disease indication and particularstatus of the subject (for example, the subject's age, size, fitness,extent of symptoms, susceptibility factors, etc), time and route ofadministration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the biologicallyactive agent(s) for eliciting the desired activity or biologicalresponse in the subject. Dosage regimens may be adjusted to provide anoptimum prophylactic or therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental sideeffects of the biologically active agent is outweighed in clinical termsby therapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of a biologically active agent withinthe methods and formulations disclosed herein is 0.01 μg/kg-10 mg/kg,more typically between about 0.05 and 5 mg/kg, and in certainembodiments between about 0.2 and 2 mg/kg. Dosages within this range canbe achieved by single or multiple administrations, including, forexample, multiple administrations per day, daily or weeklyadministrations. Per administration, it is desirable to administer atleast one microgram of the biologically active agent, more typicallybetween about 10 μg and 5.0 mg, and in certain embodiments between about100 μg and 1.0 or 2.0 mg to an average human subject. It is to befurther noted that for each particular subject, specific dosage regimensshould be evaluated and adjusted over time according to the individualneed and professional judgment of the person administering orsupervising the administration of the permeabilizing peptide(s) andother biologically active agent(s).

Dosage of biologically active agents may be varied by the attendingclinician to maintain a desired concentration at the target site. Forexample, a selected local concentration of the biologically active agentin the bloodstream or CNS may be about 1-50 nanomoles per liter,sometimes between about 1.0 nanomole per liter and 10, 15 or 25nanomoles per liter, depending on the subject's status and projected ormeasured response. Higher or lower concentrations may be selected basedon the mode of delivery, for example, trans-epidermal, rectal, oral, orintranasal delivery versus intravenous or subcutaneous delivery. Dosageshould also be adjusted based on the release rate of the administeredformulation, for example, of an intrapulmonary spray versus powder,sustained release oral versus injected particulate or transdermaldelivery formulations, etc. To achieve the same serum concentrationlevel, for example, slow-release particles with a release rate of 5nanomolar (under standard conditions) would be administered at abouttwice the dosage of particles with a release rate of 10 nanomolar.Additional guidance as to particular dosages for selected biologicallyactive agents for use can be found widely disseminated in theliterature.

Kits, packages and multicontainer units containing the above describedpharmaceutical compositions, active ingredients, and/or means foradministering the same for use in the prevention and treatment ofdiseases and other conditions in mammalian subjects are disclosedherein. Briefly, these kits include a container or formulation thatcontains one or more of the biologically active subject compoundsdescribed above formulated in a pharmaceutical preparation foradministration to a mammalian subject. The biologically active agent(s)is/are optionally contained in a bulk dispensing container or unit ormulti-unit dosage form. Optional dispensing means can be provided, forexample a pulmonary or intranasal spray applicator. Packaging materialsoptionally include a label or instruction indicating for what treatmentpurposes and/or in what manner the pharmaceutical agent packagedtherewith can be used.

The following examples are provided by way of illustration, notlimitation. These examples show that an exemplary bacterial product,Anthrax lethal toxin (LeTx) represses transactivation of the well-nownnuclear hormone receptor GR in a transient transfection system, and alsorepresses activity of an endogenous GR-regulated gene. This repressionis non-competitive and does not affect ligand binding or DNA binding,indicating that LeTx exerts its effects indirectly, presumptivelythrough a cofactor(s) involved in the interaction between GR and thebasal transcription machinery. LeTx-nuclear hormone receptor repressionis partially selective, repressing GR, and two other nuclear hormonereceptors, progesterone receptor B (PR-B) and estrogen receptor a (Era),but not the mineralocorticoid receptor (M) or ERβ. Simultaneous loss ofGR and other nuclear hormone receptor activities could render the hostmore susceptible to lethal or toxic effects of anthrax infection byremoving the normally protective anti-inflammatory effects of thesehormones, similar to the increased mortality from septic shock seen inanimals exposed to both GR antagonists and infectious agents orbacterial products. Accordingly, the present disclosure evinces for thefirst time that a bacterial product acts alters the activity of hormonereceptor. This decreased activity substantially accounts for shock andother adverse sequelae associated with bacterial infection in mammaliansubjects. More specifically, by blocking GR in the context of hostexposure to anthrax bacterial products, LeTx impairs theanti-inflammatory protective effects of glucocorticoids released duringinfection—in much the same manner as GR antagonists act in relativelyinflammatory-resistant rodents exposed to other bacterial products.

This surprising identification of nuclear hormone receptor co-factorinteractions as a mechanism of toxicity of anthrax lethal factorprovides for development of new treatments and prevention of the toxiceffects of anthrax and for novel methods and compositions to provide newand more effective tools for modulating nuclear hormone receptoractivity and diseases and other conditions mediated by diminished orexcessive levels or activity of nuclear hormone receptors and/or theircognate ligands and cofactors.

EXAMPLES Example 1 General methods

The mechanisms of action of LF inside the cell were poorly understoodprior to the present disclosure. LF is a metalloprotease that cleavesthe MAP kinase kinases (MAPKK), including MEK1, MEK2, MKK3, MKK4, MKK6and MKK7 but not MEK5 (K. R. Klimpel et al., Mol. Microbiol., 13:1093,1994; N. S. Duesbery et al., Science, 280:734, 1998; R. Pellizzari etal., FEBS lett., 462:199, 1999; R. Pellizzari et al., Int. J. Med.Microbiol., 290:421, 2000; G. Vitale et al., Biochem. J., 352,-:739,2000), thereby inhibiting the MAPK pathway. However, the fact that LeTxresistant and sensitive cells show similar internalization of LF (Y.Singh et al., J. Biol. Chem., 264:11099, 1989), and similar MPKdegradation in response to LF (R Pellizzari et al., FEBS lett., 462:199,1999; R. Pellizzari, Int. J. Med. Microbiol., 290:421, 2000), indicatesthat these factors cannot alone account for differential susceptibilityor resistance to the toxin. Other factors that have been proposed toplay a role in toxicity of LeTx include the proteosome (G. Tang et al.,Infect. Immun., 67:3055, 1999), intracellular calcium stores (S. Shin etal., Cell. Biol. Toxicol., 16:137, 2000; R. Bhatnagar et al., Infect.Immun., 57,:2107, 1989), calmodulin (R. Bhatnagar et al., Infect.Immun., 57:2107, 1989), a calyculin A sensitive protein phosphatase (J.H. Kau et al., Curr. Microbiol., 44:106, 2002), protein synthesis (R.Bhatnagar et al., Infect. Immun., 62:2958, 1994) and reactive oxygenintermediates (P. C. Hanna et al., Mol. Med., 1:7, 1994). It is notknown which of these or other unknown factors contribute to thewell-described differential cell line and rodent strain sensitivities totoxic effects of LeTx. Recently, the gene Kif1C has been determined tobe different between resistant and sensitive strains although theimplication of this is not understood (J. W. Watters et al., Curr.Biol., 11:1503, 2001; J. E. Roberts et al., Mol. Microbiol., 29:581,1998).

Fischer (F344/N) rats have long been known to be particularlysusceptible to the LeTx (F. Klein et al., J. Bacteriol., 85:1032, 1963),with death occurring within 40 minutes after exposure to a lethal dose(J. W. Ezzell et al., Infect. Immun., 45:761, 1984). F344/N rats arealso known to be relatively inflammatory disease resistant, due in partto their hypothalamic-pituitary-adrenal (HPA) axis hyper-responsivenessand resultant hyper-secretion of glucocorticoids from the adrenal glandsin response to pro-inflammmatory and other stimuli. Similar to F344/Nrats, BALB/c mice have a hyper-responsive HPA axis (N. Shanks et al.,Pharmacol. Biochem. Behav., 36:515, 1990) and are also susceptible toLeTx (S. L. Welklos et al., Infect Immun., 51:795, 1986). Ordinarilythis hyper-HPA axis responsiveness protects against inflammatory andautoimmune diseases through the anti-inflammatory and immunosuppressiveeffects of the glucocorticoids. However, F344/N rats and otherinflammatory resistant rodent strains become highly susceptible toinflammation and rapid death from septic shock after simultaneousglucocorticoid receptor (GR) or HPA axis blockade and exposure topro-inflammatory or infectious stimuli, including bacterial productssuch as streptococcal cell walls (SCW) or bacterial lipopolysaccharide(LPS) (C. K. I. Edwards et al., Proc. Natl. Acad. Sci. U.S.A., 88:2274,1991; S. H. Zuckerman et al., Infect. Immun., 60:2581, 1992; E. M.Sternberg et al., Proc. Natl. Acad. Sci. U.S.A., 86:2374, 1989; M. C.Ruzek et al., J. Immunol., 162:3527, 1999; I. A. M. MacPhee et al., J.Exp. Med., 169:431, 1989).

Cell Culture

Cos7, HTC, J774.1, Raw264.7. IC-21 and MT2 cells were grown at 37° C.and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) containing 10%serum, 10 mg/ml penicillin-streptomycin and 2 mM glutamine.

Transient Transfections

Cos7 cells were plated in 24-well plates at a density of 5×10⁵cells/well in DMEM containing 10% charcoal-stripped serum, 10 mg/mlpenicillin-streptomycin and 2 mM glutamine one day prior totransfection. Cos7 cells were transfected overnight with 20 ng receptorexpression plasmid (SVGR, ERα, ERβ, MR or PR-B), 100 ng reporterconstruct ((GRE)₂-TK luc, ERE-luc, pLTR-luc, or pGL3 control), 60 ngpSG5 (Stratagene) and 20 ng PRL TK (Promega, constitutive renillaluciferase control) using Fugene6 (Roche) according to manufacturer'sinstructions. The medium was then replaced with DMEM containing 10%charcoal-stripped serum, the appropriate hormone and LF and/or PA orinhibitor as required. After 24 hr the cells were lysed and the fireflyand renilla luciferases assayed using the dual luciferase assay(Promega).

Assay of Tyrosine Aminotransferase (TA 17 in HTC Cells

HTC cells were plated in 6 cm plates at a density of 5×10⁶ cells/platein DMEM containing 10% fetal calf serum, 10 mg/mlpenicillin-streptomycin and 2 mM glutamine one day prior to treatment.The media was then replaced with DMEM containing increasingconcentrations of dexamethasone (Dex) either alone or together withlethal factor (LF) and protective antigen (PA). After 18 hours the cellswere lysed by sonication and tyrosine aminotransferase (TAT) activityassayed as described by Thompson et al. (Proc. Natl. Adac. Sci. U.S.A.(1966) 56, 296-303).

Animal Experiments

Male and female BALB/cJ mice (10-12 weeks old, Jackson Laboratories, BarHarbor, Me.) were injected intraperitoneally (IP) with 50 mg LF, 50 mgPA, or a combination of both in 1 ml sterile-filtered phosphate bufferedsaline (PBS) 30 minutes prior to Dex treatment. Dex was injected IP in0.25 ml volume (0.06 mg/mouse). Mice were euthanized by CO₂ at varioustimes post-injection, and livers were removed, homogenized in ice-coldlysis buffer (0.2 mM pyridoxal phosphate, 0.5 mM α-keto glutarate, 0.1 Mpotassium phosphate, pH 7.6, and then centrifuged at 100,000×g at 4° C.for 30 minutes. TAT activity of supernatants was assayed as described byThompson et al. (Proc. Natl. Acad. Sci. U.S.A. (1966) 56, 296-303).

Western Blot Analysis

Cos7 cells were plated in 6-cm plates at a density of 5×10⁶ cells/platetwo days prior to treatments. Cells were treated with MAP kinaseinhibitors for 30 minutes. Cells were stimulated by addition of 10 mg/mllipopolysaccharide (LPS) or anisomycin for 30 minutes. Proteins weresolubilized using M-PER (Pierce) and 10 mg separated by sodium dodecylsulphate polyacrylamide-gel electrophoresis (SDS-PAGE) according to themethod of Laemmli (Nature (1970) 227, 680-685). Proteins weretransferred to Polyvinylidene Fluoride (PVDF) and probed with antibodiesagainst phospho-p38 MAP kinase (Thr180/Tyr182), phospho-p44/42 MAPkinase (Thr202/Tyr204) and phospho-c-Jun (Ser63) (Cell SignalingTechnology). Chemiluminescence was detected and analyzed using theChemidoc gel imaging system and volume analysis tool of the Quantity Onesoftware (Biorad).

Cytosol Prep of GR Transfected Cos7Cells

Cos7 cells were plated in 10 cm plates at a density of 1×10⁷ cells/platein DMEM containing 10% serum, 10 mg/ml penicillin-streptomycin and 2 mMglutamine one day prior to transfection. Cos7 cells were transfectedwith 2 μg SV glucocorticoid receptor (SVGR) expression plasmid usingFugene6 (Roche) according to manufacturer's instructions. After 48hours, cell cytosol was prepared by washing the cells in ice-cold PBSand then re-suspending them in ice-cold EPGMo buffer (1 mM EDTA, 20 mMpotassium phosphate pH 7.8, 10% glycerol, 20 mM sodium molybdate and 1mM DTT). The re-suspended cells were allowed to sit on ice for 10minutes and then homogenized using a glass homogenizer 30 times on ice.The broken cells were then centrifuged at 100,000×g for 3 minutes at 4°C. to pellet the cell membranes. The protein content of the supernatantcontaining the cytosol was assayed.

Gel Shift

GR gel shift oligonucleotides (Santa Cruz Biotechnology, Inc) wereallowed to anneal in a buffer containing 50 mM tris-HCl (pH 7.5-7.8), 10mM MgCl₂ and 0.1 M NaCl by heating at 65° C. for 5 minutes and thencooling slowly to room temperature. The annealed probe was thenradio-labeled with [γ-³²P]adenosine triphosphate (ATP) by incubation at37° C. for 30 minutes with T4 polynucleotide kinase (USB). The probe wasthen cleaned using a P-6 micro Bio-Spin chromatography column (Biorad)and re-suspended at a concentration of 0.5 ng/μl. The specific activityof the probe was calculated.

The binding reaction was carried out in a buffer containing 20 mM42-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.9, 20%glycerol, 100 mM KCl and 0.2 mM ethylenediaminetetraacetic acid (EDTA)with poly dI-dC, cytosol preparation, probe and competitor or LF and/orPA as required for 30 minutes at room temperature. A 40% (weight tovolume) 29:1 acrylamide/bisacrylamide Tris-Borate-EDTA (TBE) gel waspre-run in 0.5×TBE for about 20 minutes at 200 Volts. Two μl loading dyewas added to each sample before loading onto the gel. The gel was run at200 Volts until the dye front was about 1 cm from the bottom of the gel.The gel was removed, wrapped in plastic and placed against photographicfilm for autoradiography.

Ligand Binding

Ten nM [³H] dexamethasone was added to 100 μg of GR transfected cos7cell cytosol in the absence (for total binding) and presence of 500-foldexcess unlabeled dexamethasone (for non-specific binding). RU486 or LFand/or PA were added as required. The samples for incubated overnight at4° C. A sample was taken for scintillation counting. Bound ligand wasseparated from free by incubation with a 1% charcoal/0.1% dextran mixfor 10 minutes followed by centrifugation. Again, a sample was taken forscintillation counting. Specific binding was determined as totalbinding—non-specific binding.

Phospho p38 ELISA

HTC and cos7 cells were lysed with M-PER (Pierce) on ice for 30 minuteswith vortexing every 10 minutes. Phospho p38 MAPK [pTpY180/182] andtotal p38 MAPK were analyzed using phosphoELISA kits from BiosourceInternational.

Cytotoxicity Assay

Cells were plated out in a 96 well plate and incubated at 37° C. untilconfluent. The drug of interest was then added and the cells incubatedfor the required length of time. Cytotoxicity was measured using the MTTbased in vitro toxicity assay kit (Sigma) by addition of 10 μl of 5μg/ml MTT (in PBS) three hours prior to the end of the experiment. Afterthree hours the cells were lysed and the absorbance at 540 nm read.

Example 2 Lethal Toxin Repression of Dex-Induced Glucocorticoid ReceptorTransactivation in Cos 7 Cells

Cos7 cells, transiently transfected with the glucocorticoid receptor(SVGR) and a glucocorticoid response element (GRE)-luciferase reporterconstruct (GRE TK luc), were treated with 100 nM dexamethasone (Dex) andincreasing concentrations of protective antigen (PA) or lethal factor(LF) in the presence or absence of saturating concentrations of theother lethal toxin (LeTx) component. FIG. 1 shows the relativetransactivation of glucocorticoid receptor (GR) in response to 100 nMDex in the presence of various combinations of increasing concentrationsof LF, alone or together with PA. LF in the presence of 500 ng/ml PA(◯), but not alone (●), repressed GR (FIG. 1A) at concentrations as lowas 0.5 ng/ml. Also, PA in the presence of 50 ng/ml LF, but not alone,repressed GR activity at concentrations as low as 5 ng/m. Maximalrepression of GR by a combination of LF and PA at all concentrations was50%. Even in the presence of LeTx, the system can be additionally andfully repressed by co-administration of the GR/PR antagonist RU 486 withLeTx, indicating that LeTx does not prevent the action of a pureanti-glucocorticoid at the ligand binding domain.

A single amino acid substitution mutant of LF, E687C, has been shown tobe non-toxic in the LeTx sensitive macrophage cell line, RAW264.7 (K. R.Klimpel et al., Mol. Microbiol., 13:1093, 1994). This mutation has beenshown to prevent proteolytic cleavage of a peptide while still allowingLF protein to bind zinc (K. R. Klimpel et al., Mol. Microbiol., 13:1093,1994; S. E. Hammond et al., Infect. Immun., 66:23-74, 1998). In thesetransient transfection assays, in contrast to the repression induced bywild-type LF in the presence of PA (◯), the mutant LF (E687C) in thepresence of 500 ng/ml PA (□) did not repress GR (FIG. 1B). Thisindicates that this particular amino acid is important forprotein-protein interactions leading to GR repression or that theproteolytic activity of LF is required for GR repression.

Example 3 Comparison of the Effects of RU 486 and LeTX on the DoseResponse Curve of Dex in GR-Transfected Cos7 Cells

Full dose response curves of the normalized luciferase activity to Dexare shown in FIG. 2. FIG. 2A shows the effect of 500 ng/ml PA incombination with 10 ng/ml LF (●) or 50 ng/ml LF in combination with 5ng/ml PA (◯) compared to Dex alone (▪) and to Dex plus the typical GRantagonist RU 486 (□). It can be seen that either combination of LF andPA caused approximately a 50% repression of GR at all effectiveconcentrations of Dex. This pattern is indicative of a non-competitiverepressor, in contrast to a competitive antagonist, such as RU 486,which can be fully competed out at higher concentrations of Dex. FIG. 2B(insert), with data presented as a percentage of the maximal activity ineach case, shows that both combinations of LF and PA have no effect onthe EC50 value, whereas the typical competitive antagonist RU 486 causesa right shift in the curve and an increase in the EC50. Thus, thesepharmacological data indicate that LeTx represses GR activity in anon-competitive manner indicating that LeTx is not acting at theligand-binding domain of the GR

In addition, competitive ligand binding studies showed that in bothwhole cells and in cytosolic preparations neither LF nor PA, nor acombination of both, were able to compete with a saturatingconcentration of [³H] Dex for binding to GR These data, showing noeffect of LeTx on [³H] Dex binding also demonstrate that LeTx has noeffect on the number of functionally active GRs. Gel shift analysis ofGR transfected cos7 cytosol to a radiolabelled oligonucleotide showedthat LF, PA or a combination of both also had no effect on GR-DNAcomplex mobility (FIG. 8). Twenty-five μg of GR-transfected cos7 cytosolwas incubated with a [32P] labeled GRE probe in the presence of 40 foldexcess unlabeled probe as a competitor or with 5, 10 or 50 ng/ml LF, 10,50 or 500 ng/ml PA, or with 5, 10 or 50 ng/ml LF in the presence of 500ng/ml PA. The samples were run on a 40% Tris-borate-EDTA (TBE)acrylamide gel and visualized by autoradiography. The results indicatethat LeTx does not interfere with GR-DNA binding, and indicating that itacts at a point down-stream of GR-DNA binding either by interfering witha co-factor or acting itself as a co-repressor. In addition, GRtransfected cos7 cell cytosol forms a GR-GRE complex which is GRspecific as it is competed out with excess unlabeled GRE probe.Increasing concentrations of LF and PA either alone or together have noeffect on this complex indicating that LeTx does not prevent GR-DNAbinding at least in an in vitro gel shift experiments.

Example 4 Comparison of the Effects of LeTX on the Mutant 407C and WildType GR

A mutant of GR that lacks the N-terminal transactivation domain (407C)but still contains the DNA binding domain (DBD) and ligand bindingdomain (LBD), exhibits lower transactivation activity than wild type GRbut is also repressed by LeTx. At low concentrations of LeTx (0.1-0.5ng/ml LF in the presence of 500 ng/ml PA) this 407C mutant (◯) shows asmall but significantly greater repression of 1 μM dexamethasone-inducedGR activity than wild type GR (□) (FIG. 3). This indicates that LeTxacts through the DBD and/or LBD domains of GR or through pathways thatinteract with these domains of the receptor.

Example 5 LeTX Repression of Dex-Induced Tyrosine Aminotransferase (TAT)in HTC Cells

In order to determine whether the LeTx repression of GR gene activationobserved in a transient transactivation system also occurs in a morenatural system the effects of LeTx on dexamethasone induction of the GRregulated enzyme tyrosine aminotransferase (TAT) was investigated in arat hepatoma cell line (HTC-cells) (FIG. 4). HTC cells were treated for18 hours with increasing concentrations of Dex either alone (◯), ortogether with 2 ng/ml LF in the presence of 500 ng/ml PA (●) or 10 ng/mlLF in the presence of 500 ng/ml PA (▴) and TAT enzyme activity wasassayed. TAT activity was induced approximately 10-fold by Dexconcentrations as low as 10 nM. Co-treatment with either 2 ng/ml or 10ng/ml LF in the presence of 500 ng/ml PA reduced Dex induction of TATactivity by 50%, in agreement with the transient transfection assays.

Example 6 Comparison of the Effects of LeTX and PD98059 on the Responseof a Dex-Induced GRE Luciferase and a Constitutive Luciferase (pGL3)Control

Known substrates for the proteolytic action of LF include some membersof the MAP kinase family (MAPKKs). The cleavage of these proteinsresults in a blockage of the MAP kinase pathway. Cell lines exhibitingdifferential sensitivity to LeTx toxicity exhibit a similar sensitivityprofile to the MEK1 inhibitor PD98059. When the effect of this MEK1inhibitor on GR transactivation was compared to the effects of LF,PD98059 decreased luciferase activity of both the GRE-luciferase (▪) andthe constitutive luciferase vector (pGL3 control) (□) to the same extent(FIG. 5), whereas LF in the presence of PA had no effect on the pGL3control (FIG. 6).

In FIG. 5, Cos7 cells were transfected with SVGR and (GRE)₂-TK luc (▪)or with SVGR and the constitutive luciferase vector, pGL3 control(Promega) (□) and treated with 100 nM dexamethasone, and increasingconcentrations LF with 500 ng/ml PA (FIG. 5), or increasingconcentrations of the MEK1 inhibitors, PD98059 (FIG. 5B), and U0126(FIG. 5C) or the JNK inhibitor, SP600126 (FIG. 5D). Means and standarddeviations are shown and data was analyzed using a two-way ANOVAfollowed by a Scheffe post hoc test.

In FIG. 6, Cos7 cells were transfected with SVGR and (GRE)₂-TK luc (▪)or with SVGR and the constitutive luciferase vector, pGL3 control (□)and treated 100 nM dexamethasone, and increasing concentrations of thep38 MAP kinase inhibitors, SB203580 (FIG. 6A), SB220025 (FIG. 6C) andp38 MAP kinase inhibitor (FIG. 6E). Means and standard deviations areshown and data was analyzed using a two-way ANOVA followed by a Scheffepost hoc test. Cos7 cells were pre-treated for 30 min with variousconcentrations of SB203580 (FIG. 6B), SB220025 (FIG. 6D) or p38 MAPkinase inhibitor (FIG. 6F) and then further incubated with 10 μg/mlanisomycin for 30 min. Proteins were then subjected to SDS-PAGE andWestern blotting using an anti-phospho-p38 antibody.

These results show that PD98059 has a non-specific suppressive effect onluciferase, occurring through unknown mechanisms, in this transienttransfection system. Furthermore, these data show that the PD98059inhibitor does not induce any GRE-specific changes in luciferase andtherefore does not affect GR transactivation. These results alsoindicate that the mechanism of the effect of LF and PD98059 on GRtransactivation activity is different and that the LF repression of GRprobably does not occur through inhibition of the MEK1 pathway.SB203580, an inhibitor of the p38 pathway also has no effect onGR-mediated transactivation in a GRE-luciferase system.

Although LF also functions as an inhibitor of the MEK4/7 and MEK3/6pathways, this result is consistent with previous literature showingthat while activation of the MAPK pathway can repress GR, either throughactivation of ERK and JNK (M. D. Krstic et al., Mol. Biol. Cell.,17:3947, 1997; G. N. Lopez et al., J. Biol. Chem., 276:22-177, 2001; I.Rogatsky et al., Proc. Natl. Acad. Sci. U.S.A., 95:20-50, 1998), oractivation of c-Fos and c-Jun (F. C. Lucibello et al., EMBO J., 9:2827,1990; R. Schule et al., Cell., 62:12-17, 1990; P. Herrlich, Oncogene,20:24-65, 2001; M. Karin et al., J. Endocrinol., 169:447, 2001), thereis no evidence to date that a blockage of the MAPK pathway can result inGR repression.

The theory behind these experiments is that if LeTx is mediating itseffect on GR through its ability to cleave and inactivate members of theMAPK family then inhibitors of these pathways should have a similareffect in out GR transfection system. FIG. 5 shows that inhibitors ofthe MEK/ERK (PD98059 and U0126) or JNK (SP600125) pathways have no GREspecific effect. However, FIG. 6 shows that inhibitors of the p38pathway do have a repressive effect on the dexamethasone induced GRtransactivation in this system and that this repression appears to becorrelated with the inhibitors efficacy as a p38 inhibitor in thesecells. This indicates that the p38 pathway is involved.

Example 7 Effects of LeTX on Hormone-Induced Activity of Other NuclearHormone Receptors

In order to determine whether the GR repression by LeTx is specific forGR or affects other nuclear hormone receptors, transient transfectionexperiments were performed using the receptors for estrogen (ER)α, ERβ,mineralocorticoid (MR) and progesterone B (PR-B) and their respectivereporter plasmids. In contrast to its 50% repression of GR, LeTx had noeffect on MR (FIG. 7A). LeTx repressed ERα by approximately 40% (FIG.7B) but had no effect of ERβ (FIG. 7C). Finally, LeTx repressed PR-B by70% (FIG. 7D). Thus, LeTx represses nuclear hormone receptortransactivation in a partially specific manner, affecting some but notall members of this hormone receptor family.

Example 8 Evaluation of Nuclear Hormone Receptor Cofactors for theirPotential Roles in LeTx-Mediated Nuclear Hormone Receptor Repression

SRC1, TIF2 and CBP are co-factors that are known to interact directlywith the ligand binding domain (LBD) of nuclear hormone receptors suchas GR. Co-transfection of SRC1, TIF2 or CBP was undertaken according toknown methods to achieve expression of these cofactors in a suitablehost cell, and the rescue effect of this expression on LeTx-mediated GRrepression was evaluated. Co-transfection of SRC1, TIF2 or CBP todetermine whether had no-effect on LeTx repression of GR. An effect ofTIF2 alone was observed, in which this co-factor significantly enhancedthe GR transactivation. However, LeTx repressed GR transactivation40-50% in the presence or absence of TIF2. These findings indicate thatLeTx does not function directly through or prevent the action of theseco-factors. Similar to their lack of effect on GR transactivation,co-transfection of SRC1, TIF2 or CBP had no effect on LeTx repression ofPR-B. TIF2 similarly enhanced the progesterone-induced PR-Btransactivation in the absence of LeTx, and in the presence of LeTx thetoxin's 70-80% repression was maintained even with addition of 100 ngTIF2. This indicates that these co-factors are not directly involved inLeTx repression of PR-B. Additional proteins identified as cofactors(including co-activator and co-repressor proteins), as described hereinabove, will therefore be evaluated using similar cotransfection/rescueassays to determine those cofactors that are directly or indirectlyinvolved in LeTx-mediated repression of nuclear hormone receptorfunction and that will therefore provide additional screening anddiagnostic tools and therapeutic compositions and methods in accordancewith the instant disclosure.

Taken together, the foregoing examples demonstrate that LeTx repressestransactivation of both a transiently transfected and an endogenousGR-regulated gene. This repression is non-competitive and does notaffect ligand binding or DNA binding, indicating that LeTx likely exertsits effects through a cofactor(s) involved in the interaction between GRDBD/LBD and the basal transcription machinery.

LeTx exhibits a maximum of 50% repression of GR and 70% repression ofPR-B. Such partial repression is indicative of the target of LeTx beingdown-stream of GR-DNA binding in the interaction of GR with the basaltranscription machinery. As there are multiple proteins involved in thisinteraction, if one component is removed and/or repressed, thenremaining, intact co-factors could still allow some but not fullactivity of the receptor.

The ability of LeTx to repress the 407C mutant GR, which lacks theN-terminal transactivation domain, indicates that proteins that interactwith the DBD and/or LBD of GR are involved directly or indirecely inthis repression. The small but significantly greater repression at lowconcentrations of LeTx indicates that the N-terminal transactivationdomain of wild-type GR may be slightly protective of this repression.

Contrary to previous models proposed in the literature, the MEK1 pathwayis probably not involved in LeTx activity. The MEK1 inhibitor PD98059did not alter GR repression in a transient transfection assay. Thespecificity of repression of some but not all members of the nuclearhormone receptor family tested also supports the notion that LeTx isworking through a co-factor rather than through a direct interactionwith the GR receptor.

In light of the foregoing description, LeTx repression of nuclearhormone receptors in vivo in the course of anthrax infection likelycontributes to some of the adverse symptoms of anthrax. Since theglucocorticoid receptor is essential for survival and also formodulation of immune responses to infectious agents, inhibition ofglucocorticoid receptor activity during infection is proposed to renderthe host more susceptible to the lethal or toxic effects of anthraxbacteria. Simultaneous loss of activity of other nuclear hormonereceptors, particularly PR, would potentially amplify these immuneenhancing effects. Indeed, this scenario is consistent with thewell-described increased mortality from septic shock in rodents thathave been adrenalectomized or treated with the GR/PR receptor antagonistRU 486, and simultaneously exposed to infectious agents orpro-inflammatory bacterial products (C. K. I. Edwards et al., Proc.Natl. Acad. Sci. U.S.A., 88:2274, 1991; E. M. Sternberg et al., Proc.Natl. Acad. Sci. U.S.A., 86:2374, 1989; M. C. Ruzek et al., J. Immunol.,162:3527, 1999; I. A. M. MacPhee et al., J. Exp. Med., 169:431, 1989).The GR repression by LF could also contribute to the long-terminflammatory and fatigue sequelae now being reported in relation toanthrax exposure (J. A. Jernigan et al., Emerg. Infect. Dis., 7:933,2001), since blunted glucocorticoid responses have been associated withmany inflammatory diseases and fatigue states (G. Neeck et al., Rheum.Dis. Clin. North Am., 26; 989, 2000). Application of the compositionsand methods provided herein to further map nuclear hormone receptorco-factor interactions as a mechanism of in vivo action of anthrax LFwill thus yield important new tools for treatment and prevention of theadverse effects of this toxin and other bacterial products havingsimilar activities.

In accordance with the foregoing results and additional teachingsherein, additional, confirming studies will be undertaken to identifymore specific aspects of the subject technology, in particular morespecific aspects of the molecular mechanism(s) of LF/PA effect on GR andother nuclear hormone receptors. Certain molecular studies will focus onelucidating the precise molecular mechanism(s) by which LeTx interactswith and represses GR and other nuclear hormone receptors; determiningwhether LeTx interacts with a GR and other nuclear hormone receptorco-factors or acts as a co-repressor itself; and determining whetherLeTx can affect GR and other nuclear hormone receptor gene repression aswell as gene activation.

Example 9 Effect of LeTx on GR Gene Repression

Since the mechanism of GR repression and activation of genes differs,LeTx also may affect GR-mediated gene repression in addition to therepression described in the foregoing examples. In order to elucidatethese further aspects of the disclosure, transient transfectionexperiments comparable to those presented above are performed usingcells transfected with known vectors encoding NFκB or AP-1 and theirrespective reporter constructs, together with increasing concentrationsof GR. Cells are then treated with appropriate ligand for NκB and AP-1and Dex together with increasing concentrations of LeTx. GR generepression is measured in the luciferase reporter system as describedfor the GRE-reporter.

Example 10 Identification of Co-Factors Involved in LeTx Effect

In order to identify which co-factors (co-activators/co-repressors) areaffected by LeTx, and to determine if the GR repression by LeTx can beovercome by supplementation with such co-factors, key members of each ofthe major families of cofactors (SRC-1, TIF2, pCIP (AIB1), CBP and pCAF)are co-transfected in increasing amounts into the GR/GRE transientlytransfected Cos 7 cells and GR activation is measured in theGRE-luciferase transactivation assay in the presence and absence of arange of doses of LF and PA alone or together. Expression plasmids for alarge panel of cofactors are readily obtained from academic,institutional and commercial sources in the art, and these expressionvectors can be readily utilized in transient transfection and relatedassays available in the art. A large number of cofactors can beevaluated by these assays, including well-known high throughput assays,for use within the methods and compositions of the disclosure. Among thesubject cofactors for use within these screening aspects of thedisclosure are those listed in the exemplary listing provided in Table 2above.

Thus, a method is provided for identifying a nuclear hormone receptorcofactor that is an agonist or antagonist of a selected nuclear hormonerecepetor. The method includes the steps of:

-   -   (1) providing a viable test cell that expresses the cofactor and        the nuclear hormone receptor, and a substrate/reporter construct        for the nuclear hormone receptor, wherein expression of the        substrate reporter construct is detectable and provides a        measurement of nuclear hormone receptor pathway activity;    -   (2) providing a viable control cell that expresses the nuclear        hormone receptor and the substrate/reporter construct for the        receptor but has reduced or no expression of the cofactor in        comparison to cofactor expression in the test cells;    -   (3) contacting the test and control cells with a bacterial        product that modulates the nuclear hormone receptor pathway;    -   (4) detecting and comparing nuclear hormone receptor pathway        activity between the test and control cells to determine whether        the cofactor enhances or impairs modulation of the receptor        pathway activity by the bacterial product.

Example 11 Dissection of Region of GR Involved in LeTx Repression

The region of GR required for LF/PA repression is defined according toknown methods using mutant and chimeric forms of the GR. The mutant andchimeric constructs are transiently transfected into Cos 7 cells in atransient transfection assay as described above. Activation of the GR isassessed in the presence and absence of a range of doses of LF and PAalone or together. In exemplary embodiments, several available mutantslacking specific regions of GR, and known chimeras of PR/GR and MR/GRare used. These include, for example: 407C—lacks a transactivationdomain, contains DBD and LBD of GR (D. Szapary et al., J. Biol. Chem.,271 :30576-82, 1996), GR/PR—transactivation domain and DBD of GR andhinge and LBD of PR; PR/GR—transactivation domain and DBD or PR andhinge and LBD of GR (L. N. Song et al., J. Biol. Chem., 276:24806-16,2001); MR/GR chimeras containing the N-terminal domain of GR and the DBDand LBD or MR and vice versa.

Example 12 Interactions Between LF/PA and Components of the GR-GREComplex

Gel shift analyses have not shown an effect of LF/PA on the GR-GREcomplex. This in vitro system indicates that LeTx does not interactdirectly with the GR-GRE complex, as it does not further shift thisband. However, there are many proteins in the GR transactivationcomplex, downstream of GR-GRE, with which LF/PA may interact to affectGR responses without any direct interaction with GR-GRE. Therefore, inorder to elucidate direct interactions between LF/PA and identifiablecomponents of the GR-GRE complex or co-factors, co-immunoprecipitationstudies are performed using available polyclonal and monoclonalantibodies to LF. In these assays, OR is obtained from cell lysates anddexamethasone and LF+PA is added to the mixture. A parallel set ofexperiments is also performed using whole cells. Known proteins (LF, GR,MAPK) are identified in gel shift assays, for example, by Westernblotting. Unknown proteins in the complex are identified according towell-known methods (for example, mass spectrometry). Ifco-immunoprecipitation is insensitive, GST (glutathione-S-transferase)LF pull-downs are performed to identify whether any direct interactionswith any components of the GR complex occur.

Example 13 Response of Endogenous GR-Regulated Genes to LeTx

The effects of LeTx on expression of endogenous genes known to beinduced or repressed by glucocorticoids are further assessed in intactcell lines and primary cell cultures. Genes known to be repressed by GRinclude, for example, IL-6, TNFα, collagenase and COX-2, via the NFκBand AP-1 pathways. Genes known to be activated by GR includemetallothionein IIa, tyrosine amino transferase (TAT),phosphoenolpyruvate carboxykinase (PEPCK) and glutamine synthase (GS).In these assays, mRNA, protein expression or enzyme activity ofdexamethasone regulated genes is measured according to conventionalmethods in cell lines that contain endogenous GR but in which LeTx isnon-toxic.

Example 14 Additional Nuclear Hormone Receptors and Domains

To identify other nuclear hormone receptors modulated by LeTx and otherbacterial products, transient transfection systems as outlined above areemployed. For example, the effect of LeTx and other bacterial products(for example, as identified in Table 1 above) on a panel of nuclearhormone receptors, including androgen receptor (AR), mineralocorticoidreceptor (MR), progestin receptor (PR), estrogen receptor (ER), thyroidhormone receptor (TR), vitamin D receptor (VDR), retinoid receptor (RARor RXR), peroxisome receptor (XPAR or PPAR), icosanoid receptor (IRs),and orphan receptors, for example steroid receptor and thyroid receptor.

In one embodiment, a method is disclosed that is a method of identifyinga domain or amino acid sequence motif of a nuclear hormone receptorinvolved in modulation of activity of the nuclear hormone receptor by abacterial product. The method includes the steps of providing a viabletest cell that expresses a mutant, chimeric, or truncated form of thenuclear hormone receptor and a substrate/reporter construct for thenuclear hormone receptor, wherein expression of the substrate reporterconstruct is detectable and provides a measurement of nuclear hormonereceptor pathway activity; providing a viable control cell thatexpresses a full-length or functionally wild type nuclear hormonereceptor and the substrate/reporter construct for the receptor;contacting the test and control cells with a bacterial product thatmodulates the wild type nuclear hormone receptor pathway; and detectingand comparing nuclear hormone receptor pathway activity between the testand control cells to determine whether the cofactor enhances or impairsmodulation of the receptor pathway activity in the cells expressing themutant, chimeric, or truncated form of the nuclear hormone receptor.Thus, a determination is made whether structural elements present in themutant, chimeric, or truncated form of the receptor are involved inmodulation of activity of the nuclear hormone receptor by a bacterialproduct by the bacterial product. In some embodiments of the method, thebacterial product is a bacterial toxin, for example, anthrax lethalfactor (LF) or lethal toxin (LeTx). In particular examples, the nuclearhormone receptor is selected from glucocorticoid receptor (GR),progestin receptor (PR), and estrogen receptor-α (ER-α).

Example 15 In Vivo and Clinical Relevance of Nuclear Hormone ReceptorRepression by LeTx

To further elucidate the clinical significance of LeTx-GR/nuclearhormone receptor interactions as they relate to inflammation,autoimmunity, toxicity and lethality associate with anthrax and otherbacterial diseases, and their cognate vaccines, the following studiesare performed. Attendant goals in this context include:

i. To determine in vitro whether macrophages from rat strainsdifferentially susceptible to anthrax LeTx, or cell lines that differ insusceptibility and resistance to anthrax LeTx show differences in GRnumber, affinity, function or cytotoxicity to GR antagonists.

ii. To elucidate how nuclear hormone receptor repression mediated bybacterial products alters inflammation, autoimmunity, toxicity andlethality associate with anthrax and other bacterial diseases, and theircognate vaccines.

iii. To identify other bacterial toxins that act as GR and/or othernuclear hormone receptor repressors.

Example 16 In Vitro Cell Culture Studies of Macrophage GR Number andFunction in LeTx Resistant and Susceptible Macrophages

Several macrophage cell lines exist that are relatively sensitive(J744.1 and RAW264.7) or resistant (IC-21 and MT-2) to cytotoxicityafter exposure to LeTx. Since MAPKK degradation by LeTx does not differin these sensitive and resistant cells lines, an additional factor(s)must contribute to their differential sensitivity. GR number, bindingcharacteristics and function in these cell lines are evaluated in orderto further define the contribution of endogenous differences in GRfunction in this differential sensitivity. While a lack of difference inGR function does not rule out the involvement of GR or its co-factors inLeTx differential toxicity, small differences in GR function, compoundedby LeTx GR repression, may account for such differences.

GR number and function of peritoneal macrophages from F344/N and LEW/Nrats are evaluated in parallel, since F344/N rats are more susceptibleto the lethal effects of in vivo administered LeTx than are LEW/N rats.GR number and affinity are readily measured, for example, usingradiolabeled ³H-Dex in ligand binding assays. Function is assessed, forexample, by evaluating endogenous GR activated or GR repressed genes, asdescribed above.

In addition, it will be determined whether the GR/PR ligand-bindingantagonist RU486 is differentially toxic to, or reverses the sensitivityand resistance of, macrophages to LeTx. In these assays, RU486 is addedalone or together with LeTx in varying doses to sensitive and resistantcell lines and cytotoxicity are measured, for rexample, in a standardMTT cytotoxicity assay (Sigma, Mo.)

Finally, expression of other factors identified through theabove-described molecular studies are evaluated and quantified, andfurther assays developed to reconstitute missing/defective factor(s) todetermine whether nuclear hormone receptor repression by LeTx and otherbacterial products can be overcome by such replacement.

Example 17 Int vivo GC Antagonism by Bacterial Products and HPA Axis

(a) Differential Pre-Morbid HPA Axis Responsiveness and DifferentialStrain Susceptibility to Anthrax Lethality:

To evaluate clinical aspects of the disclosure, for example howpre-morbid HPA axis responsiveness is associated with differentialstrain susceptibility to anthrax, clinical effects on blood pressure,heart rate and temperature, chronic inflammation, autoimmune effects,and lethality are assessed according to various protocols. For example,hyper-HPA axis responsive F344/N rats and hypo-HPA axis responsive LEW/Nrats are employed as test subjects. If differential responses are found,both strains of rats are treated with Dexamethasone (Dex) to determinewhether Dex replacement overcomes or prevents the symptoms. However, asin vitro studies indicate that LeTx is an irreversible GC repressor, itis unlikely that Dex would prevent or overcome the toxic effects ofLeTx. Testing of agents to counter the effects of LeTx is informed bythe outcome of in vitro molecular mechanism studies.

(b) LeTx Acute Effects In Vivo on LPS-Induced Inflammatory Responses,Septic Shock and HPA Axis Responses:

To further evaluate how LeTx acts as a GR antagonist in vivo, F344/Nrats are treated with bacterial lipopolysaccharide (LPS) as a stimulusto the HPA axis at the same time as a range of sub-lethal doses of LeTxare administered intra-peritoneally as a GR antagonist. Studies areperformed as previously described for SCW and RU486 experiments (E. M.Sternberg et al., Proc. Natl. Acad. Sci. U.S.A., 86:2374-8, 1989).Plasma levels of corticosterone, plasma cytokines that are usuallyreleased during septic shock (TNF-α, IL-6 and IL-1), as well as bloodpressure, heart rate and temperature are monitored at different timepoints prior to and after treatment over a one hour period. Mortality indifferent groups is recorded. In these assays, LeTx antagonism of GR andPR is predicted that it might have a similar effect as other GR/PRantagonists for example, RU 486, leading to rapid death from septicshock by blocking the anti-inflammatory effects of glucocorticoids.Plasma corticosterone and ACTH responses are predicted to increase,depending on the degree to which LeTx blocks glucocorticoid negativefeedback of the HPA axis and peripheral cytokine production. LeTxblockade of the effects of GC in suppressing the HPA axis, plasma Cortand ACTH are expected to increase, resulting in a situation of highplasma Cort and relative peripheral GC resistance.

(c) Ex Vivo Measurement of GR-Mediated Gene Induction and GeneRepression.

Glucocorticoid (GC) sensitivity is assessed using a whole blooddexamethasone induction and LPS-stimulation/GC-suppression assaypreviously described (R. H. DeRijk et al., Journal of ClinicalEndocrinology and Metabolism, 81:228-35, 1996). Whole blood is also bestimulated ex vivo with LPS, and cytokine production in supernatants ismeasured in the presence and absence of varying doses of dexamethasone(Dex) +/−LF +/−PA. RU486 in varying doses is used as a positive control.RU486 blocks the glucocorticoid receptor, thereby preventing the Dexsuppression of LPS-induced cytokines. Concentrations of cytokines insupernatants are measured by ELISA and/or by immunoaffinity capillaryelectrophoresis (T. M. Phillips et al., Electrophoresis, 19:2991-6,1998).

(d) In Vivo Co-Factor Effects on LeTx Toxicity:

In conjunction with the above-described molecular studies, and in orderto further assess how co-factors identified as targets of LeTx in vitrooperate in LeTx's toxicity in vivo, knock-out mice not expressing theidentified co-factor(s) and transgenic mice over-expressing theidentified co-factor(s) are studied. The animals are treated with arange of doses of LeTx and HPA axis responses, cytokine and bloodpressure, temperature, and any other sickness responses are compared towild-type. In addition, the effects of LeTx are tested in GRdimerization mutant (GR^(dim/dim)) mice, in which GR gene repressionoccurs, but GR gene activation does not occur. A range of concentrationsof LeTx or vehicle control is administered to knock-out and wild-typecontrols and HPA axis responses, cytokine and blood pressure,temperature and any other sickness responses are compared to wild-type.GR^(dim/dim) dim mice are obtained from Dr. Jan-Ake Gustafsson,Karolinska Institute, Stockholm, Sweden.

(e) Chronic In Vivo Effects of LeTx on Inflammatory/Autoimmune Diseasein Animal Models:

The manner and mechanisms by which LeTx operates in widely acceptedmodels of inflammatory and autoimmune diseases is assessed. LEW/N andF344/N rats are injected subcutaneously with complete Freund's adjuvantas previously described (Webster et al., J. Rheumatol. 29:1252-61, 2002)and LeTx or vehicle control are simultaneously administeredintraperitoneally in sub-lethal doses selected from pilot studies usingLeTx alone. Rats are scored daily for four weeks for arthritis severity(arthritis index) and body weight, as previously described. At the endof this period a full autopsy is performed, and tissues, includingsynovial tissue, are analyzed for evidence of inflammation.

In addition, another model for use within these aspects of thedisclosure is the model of relapsing murine experimental allergicencephalomyelitis induced by myelin basic protein (the EAE model). Thiswidely accepted animal model for evaluating treatments for multiplesclerosis is described, for example, in Fritz et al., J. Immunol. 130:1024-6, 1983. A related model has been described using rat subjects byMacPhee et al., J. Exp. Med. 169:43145, 1989. In this model, EAE isinduced in Lewis rats and causes paralysis. Endogenous glucocorticoidsameliorate the effects of EAE. Adrenalecomized rats were implanted witha coritcosterone pellet. If it mimicked the basal GC levels, then theanimals died. If it mimicked the GC levels during the EAE disease, thenthe animals survived and the level of disease was comparable tonon-adrenalectomized animals. If the GC levels were higher then diseaseremission was achieved. These models are therefore useful in the contextof assays to evaluate the clinical significance and mechanisms ofbacterial product suppression of GR and associated impacts on autoimmunediseases.

Example 18

PA and/or LF Do Not Prevent [³H] dexamethasone Binding to GR TransfectedCos7 Cell Cytosol Preparations

This example demonstrates that PA and/or LF do not prevent [³H]dexamethasone binding to GR transfected cos7 cell cytosol preparations.One hundred μg GR transfected cos7 cytosol was incubated overnight with10 nM [³H] dexamethasone in the presence or absence of 500 fold excessunlabeled dexamethasone and in the presence of 1 μM RU486, 500 ng/ml PA,50 ng/ml LF or 500 ng/ml PA+50 ng/ml LF. Bound was separated from freeand specific binding calculated. The percent specific binding incomparison to dexamethasone alone is shown (FIG. 9). These results showthat RU486 can compete with a saturating concentration of 3Hdexamethasone where as PA, LF or LF+PA cannot. Therefore LeTx does notfunction as a normal GR antagonist such as RU486 in that it does notcompete with dexamethasone for ligand binding. Also, if there were adecrease in the number of glucocorticoid receptors one would expect theamount of saturating ³H dexamethasone binding to decrease. Therefore,LeTx does not effect the number of glucocorticoid receptors. This resulthas been confirmed with Western blotting.

Example 19 RU486 Can Fully Repress Dexamethasone-Induced GRTransactivation and Progesterone-Induced PR-B Transactivation in Cos7Cells Even in the Presence of LeTx

This example demonstrates that RU486 can fully repressdexamethasone-induced GR transactivation and progesterone-induced PR-Btransactivation in cos7 cells even in the presence of LeTx. Cos7 cellswere transfected with SVGR and (GRE)₂-TK luc or PR-B and pLTR luc andthen treated with 100 nM dexamethasone or progesterone in the presenceof 2 ng/ml LF+500 ng/ml PA and increasing concentrations of RU486(maximum 1 μM). Relative luciferase values were measured (FIG. 10).These results show that addition of RU486 in combination with LeTxallows full repression of both GR and PR-B. This indicates thattranscription can be fully repressed in the presence of LeTx.

Example 20 Over Expression of TIF2 Does Not Overcome LeTx Repression ofDexamethasone-Induced GR Transactivation

This example demonstrates that over expression of TIF2 does not overcomeLeTx repression of dexamethasone-induced GR transactivation. Cos7 cellswere transfected with SVGR and (GRE)₂-TK luc and increasing amounts ofTIF2 expression plasmid (maximum 100 ng) and then treated with 100 nMdexamethasone in the presence of 2 ng/ml LF+500 ng/ml PA or 10 ng/mlLF+500 ng/ml PA. Relative luciferase values were measured. Relative foldinduction and percent repression by LeTx are shown. If LeTx is removingone of the many cofactors involved in the interaction between theGR/PR-B and the transcriptional machinery, then over-expression of thisfactor may overcome the repression. CBP, SRC-1 and TIF2 are the majorcofactors that are known to interact directly with these nuclear hormonereceptors. Over-expression of these (CBP, TIF2 and SRC-1) was unable toovercome the repression of GR or PR-B by LeTx.

Example 21 LeTx Repression of Dexamethasone Induced TyrosineAminotransferase (TAT) in Mouse Livers

This example demonstrates that LeTx repression of dexamethasone inducedtyrosine aminotransferase (TAT) in mouse livers. BALB/cJ mice wereinjected with LeTx and 30 minutes later with Dex. After six and twelvehours liver TAT activity was assayed (FIG. 11). Means and standarddeviations of six to ten animals are shown and a two-way ANOVA followedby a Scheffe post hoc test was performed. These results show that LeTxis able to also repress dexamethasone induction of tyrosineaminotransferase (TAT) activity in mouse livers.

Example 22 MAPK Inhibitors Repress Dexamethasone-Induced TAT Activity inHTC Cells

This example demonstrates that MAPK inhibitors repressdexamethasone-induced TAT activity in HTC cells. HTC cells were treatedwith dexamethasone either alone (dex) or together with 2 ng/ml LF+500ng/ml PA, 50 μM PD98059, 50 μM U0126, 50 μM SP600125, 50 μM SB203580 or20 μM SB220025 for 18 hr and TAT activity assayed. MAPK inhibitors weretested to determine whether they were able to repress dexamethasoneinduction of TAT in HTC cells. They did have an effect in on TATinduction, although this system cannot distinguish between GR specificand non-specific effects as the cos7 cells. However, these resultsindicate that the p³⁸ repression of GR is not an artifact due to thecos7 cells, but also occurs in these HTC cells.

Example 23 LeTx Inhibits Endogenous Phospho P38 in GR Transfected Cos7Cells and HTC Cells

This example demonstrates that LeTx inhibits endogenous phospho P38 inGR transfected cos7 cells and HTC cells. Phospho P38 (FIGS. 14 A and 14B) and total P38 (FIGS. 14C and 14D) were measured by phosphoELISA insamples of GR transfected cos7 cells (FIGS. 14A and 14C) or HTC (FIG.14B or 14D) treated with 100 nM Dexamethasone and increasingconcentrations of LF in the presence of 500 ng/ml PA.

This experiment was designed to determine the relative content of p38and phospho p38 in cos7 and HTC cells treated with dexamethasone andLeTx. These results show that LeTx does indeed repress p38 (as shown bya decrease in phospho p38) in the LeTx experiments. Thus, together withthe data in Example 22, this example shows that during out LeTxrepression of GR experiments in both cos7 and HTC cells the LeTx alsorepresses P38. In addition, inhibition of p38 correlates with repressionof GR.

Example 24 J774.1 and Raw264.7 Macrophage Cell Lines are Sensitive toLeTx Whereas IC-21 and MT2 Macrophage Cell Lines are RelativelyResistant

This example demonstrates that J774.1 and Raw264.7 macrophage cell linesare sensitive to LeTx whereas IC-21 and MT2 macrophage cell lines arerelatively resistant. J774.1, Raw264.7, IC-21 and MT2 cells were grownin DMEM and exposed to increasing concentrations of LF in the presenceof 500 ng/ml PA for 24 hours. MTT assay was performed at the end of the24 hours and the percent cell survival is shown as the percentage cellssurviving compared to cells that have not been exposed to LeTx. Thus,there exist macrophage LeTx sensitive and resistant cell lines.

Example 25 Pretreatment of Dexamethasone or RU486 Does Not Prevent LeTxToxicity in J744.1 or Raw264.7 Cell Lines

This example demonstrates that pretreatment of dexamethasone or RU486does not prevent LeTx toxicity in J744.1 or Raw264.7 cell lines. J774.1and Raw264.7 cells were grown in DMEM and exposed to increasingconcentrations of LF or LFm (E687C) in the presence of 500 ng/ml PA for24 hours. In some cases the cells were pre-treated with 100 nMdexamethasone or 0.2 μM or 1 μM RU486 for 2 hours prior to LeTxtreatment. MTT assay was performed at the end of the 24 hours and thepercent cell survival is shown as the percentage cells survivingcompared to cells that have not been exposed to LeTx. Thus, co treatmentwith with dexamethasone or RU486 has no effect on the LeTx cytotoxicityof the sensitive cell lines.

Example 26 Rolipram Does Not Prevent LeTx Repression ofDexamethasone-Induced TAT Activity in HTC Cells

This example demonstrates that rolipram does not prevent LeTx repressionof dexamethasone-induced TAT activity in HTC cells. HTC cells weretreated with 1 or 10 μM dexamethasone and/or 10 μM rolipram either alone(treatment) or together with 2 ng/ml LF+500 ng/ml PA or 10 ng/ml LF+500ng/ml PA for 18 hours and TAT activity assayed. A drug that activates GRand circumvents the point at which LeTx represses GR has the potentialas use as a therapeutic. One such drug is rolipram, which is aphosphodiesterase inhibitor but has been show to activate GR. These datashow that rolipram is unable to prevent the LeTx repression of dexinduced TAT activity in HTC.

Example 27 Rolipram Does Not Prevent LeTx Toxicity in Raw264.7 CellLines

This example demonstrates that rolipram does not prevent LeTx toxicityin Raw264.7 cell lines. Raw264.7 cells were grown in DMEM and exposed toincreasing concentrations of LF in the presence of 500 ng/ml PA for 24hours. In some cases the cells were pre-treated for two hours,co-treated or pre- and co-treated with 10 μM rolipram. MTT assay wasperformed at the end of the 24 hours and the percent cell survival isshown as the percentage cells surviving compared to cells that have notbeen exposed to LeTx. Thus, rolipram is unable to prevent LeTxcytotoxicity in a sensitive macrophage cell line.

Example 28 The Extent of LeTx Repression of Progesterone-Induced GR,PR-B and GR/PR Chimera Transactivation in cos7 Cells is Dependent on thePromoter Construct

This example demonstrates that the extent of LeTx repression ofprogesterone-induced GR, PR-B and GR/PR chimera transactivation in cos7cells is dependent on the promoter construct. Cos7 cells weretransfected with the receptor expression plasmids for GR, PR-B, and thetwo chimeras GR/PR and PR/GR, and with the reporter constructs (GRE)₂ TKluc (solid symbols) or PLTR luc (open symbols) and subsequently treatedwith 100 nM progesterone and increasing concentrations of LF in thepresence of 500 ng/ml PA. Relative luciferase induction is shown.

LeTx represses GR transactivation on a (GRE)2TK luc promoter by 40-50%and represses PR-B transactivation on a pLTR-luc promoter by 70% (seeabove). This difference was examined using chimeras of GR/PR andchanging the promoters, since both GR and PR-B are able to activate bothof these promoter constructs. These data show that the difference inrepression (40-50% versus 70% repression) is a function of the promotercontext. All of the receptors repress the pLTR-luc promoter to a greaterextent than the (GRE)2TK luc promoter.

Example 29 The Extent of LeTx Repression of Dexamethasone-Induced GR andGR/PR Chimera Transactivation in cos7 Cells is Dependent on the PromoterConstruct

This example demonstrates that the extent of LeTx repression ofDexamethasone-induced GR and GR/PR chimera transactivation in cos7 cellsis dependent on the promoter construct. Cos7 cells were transfected withthe receptor expression plasmids for GR, and the two chimeras GR/PR andPR/GR, and with the reporter constructs (GRE)₂ TK luc solid symbols) orPLTR luc (open symbols) and subsequently treated with 100 nMDexamethasone and increasing concentrations of LF in the presence of 500ng/ml PA. Relative luciferase induction is shown.

LeTx represses GR transactivation on a (GRE)2TK luc promoter by 40-50%and represses PR-B transactivation on a pLTR-luc promoter by 70% (seeabove). This difference was examined using chimeras of GR/PR andchanging the promoters, since both GR and PR-B are able to activate bothof these promoter constructs. These data show that the difference inrepression (40-50% versus 70% repression) is a function of the promotercontext. All of the receptors repress the pLTR-luc promoter to a greaterextent than the (GRE)2TK luc promoter.

Example 30 Extent of LeTx Repression of Aldosterone-, Corticosterone,and Dexamethasone-Induced GR/MR Chimera Transactivation in Cos7 Cells

This example demonstrates the extent of LeTx repression of aldosterone-,corticosterone, and dexamethasone-induced GR/MR chimera transactivationin cos7 cells. Cos7 cells were transfected with the receptor expressionplasmids for GR, MR and various GR/MR chimeras and with the reporterconstructs (GRE)₂ TK, luc (FIGS. 21A, 21C, and 21E) or pltruc (FIGS. 21Band 21D) and subsequently treated with 100 nM aldosterone (FIGS. 21A and21B), 1 μM corticosterone (FIGS. 21C and 21D), or 100 nM dexamethasoneand increasing concentrations of LF in the presence of 500 ng/ml PA.Relative luciferase induction is shown.

The rational behind this example is that using chimeras of MR and GR onthe (GRE)2TK promoter will help us determine which region of the GR isrequired for the repression. This indicates that the end of theN-terminal domain and the DNA binding domain (amino acids 404-525) isrequired for LeTx repression.

Example 31 Method for Diagnosis

A method for diagnosis of a subject having or at risk of having adisorder associated with a cofactor of a nuclear hormone receptor isdisclosed herein. The disorder can be associated with an increase or adecrease in the cofactor of the nuclear receptor, as compared to asubject not affected by the disorder. In one embodiment, the method isused to identify an individual at risk for toxic effects of exposure topathogenic bacteria, for example anthrax. The method includes obtaininga sample from the subject that includes a cofactor of a nuclear hormonereceptor. The sample is contacted with the bacterial product. Anincrease in the binding of the bacterial product indicates that theco-factor is increased as compared to a normal subject (a subject notaffected with the disorder). A decrease in the binding of the bacterialproduct indicates that the co-factor is decreased as compared to anormal subject. Thus, the binding of bacterial product to the sampleindicates that the subject has the disorder.

In one example, the bacterial product is directly labeled. In anotherexample, an antibody is utilized that specifically binds the bacterialproduct. These antibodies are of use, for example, in immunoassays inwhich they can be utilized in liquid phase or bound to a solid phasecarrier. In addition, the antibodies in these immunoassays can bedetectably labeled in various ways. Examples of types of immunoassayswhich can utilize antibodies are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the bound bacterial product using theantibodies can be carried out utilizing a variety of immunoassays,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

In one example, the bacterial product is LeTx or LF, and the disorder isassociated with increased or decreased expression of GR, PR, or β-ER.Certain strains of rodents show enhanced susceptibility to lethaleffects of exposures to anthrax. As demonstrated herein, differences incharacteristics of a nuclear hormone receptor in these animals isindicative that they are susceptible to anthrax. Thus, in order todetermine if an individual is highly susceptible or highly resistant toan anthrax infection, a sample can be obtained from the individual thatincludes nuclear hormone receptors. The sample is contacted with LeTx orLF, and the binding of the toxin to the sample is assessed. A change inthe binding of LeTx or LF, as compared to a normal subject, can be usedto demonstrate that the subject is either highly susceptible or highlyresistant to an anthrax infection.

Although the foregoing disclosure has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications may be practicedwithin the scope of the appended claims which are presented by way ofillustration not limitation. In this context, various publications havebeen cited within the foregoing disclosure for economy of description

1. A method for identifying an agent that modulates the activity of anuclear hormone receptor comprising the steps of: providing a viablecell that expresses a nuclear hormone receptor and a nuclear hormonereceptor substrate, reporter construct, or both, wherein expression ofthe substrate reporter construct is detectable and provides ameasurement of nuclear hormone receptor pathway activity; contacting atest cell with a test agent and an isolated bacterial product;contacting a control cell with the isolated bacterial product in theabsence of the agent; detecting and comparing nuclear hormone receptoractivity between the test and control cell to identify a test agent thatinteracts with the nuclear hormone receptor and modulates the activityof the nuclear hormone receptor by the bacterial product.
 2. The methodof claim 1, wherein the nuclear hormone receptor is selected from aglucocorticoid receptor (GR), androgen receptor (AR), mineralocorticoidreceptor (MR), progestin receptor (PR), estrogen receptor (ER), thyroidhormone receptor (TR), vitamin D receptor (VDR), retinoid receptor (RARor RXR), peroxisome receptor (XPAR or PPAR), icosanoid receptor (IRs),steroid receptor and thyroid receptor.
 3. The method of claim 2, whereinthe nuclear hormone receptor is GR.
 4. The method of claim 2, whereinthe nuclear hormone receptor is PR.
 5. The method of claim 1, whereinthe bacterial product is a bacterial wall protein, soluble bacterialprotein, or lipopolysaccharide.
 6. The method of claim 1, wherein thebacterial product is a bacterial toxin that is not endotoxin.
 7. Themethod of claim 6, wherein the bacterial toxin elicits one or moresymptoms of a toxic effect, inflammatory response, stress, shock,chronic sequelae, autoimmunity, or mortality in a susceptible hostinfected with a bacterium that produces the toxin.
 8. The method ofclaim 6, wherein the bacterial toxin exhibits metalloprotease activity.9. The method of claim 8, wherein the bacterial toxin is anthrax lethalfactor (LF) or lethal toxin (LeTx) or a metalloenzyme of Clostridiumtetanus or C. botulinum bacteria.
 10. The method of claim 1, wherein thebacterial product is a bacterial antigen.
 11. The method of claim 10,wherein the bacterial antigen is a pyrogenic toxin superantigen (PTSAg).12. The method of claim 1, wherein the agent exerts its effect on thenuclear hormone receptor is through a mechanism other than inhibition ofa MEK1 or MAPKK pathway.
 13. The method of claim 1, wherein the agent isa genetically engineered or chemically modified variant or mimetic ofthe bacterial product, a drug, or a cofactor for the nuclear hormonereceptor.
 14. The method of claim 1, wherein the agent is effectivefollowing administration to a mammalian subject to reduce one or moreinflammatory and/or autoimmune symptoms that can accompany exposure tothe bacterial product or infection by a pathogen expressing the product.15. The method of claim 1, wherein the isolated bacterial product altersthe activity of the nuclear hormone receptor and does not alter numberof nuclear hormone receptors on the viable cell.
 16. A method foridentifying an agent that inhibits nuclear hormone receptor repressionby a bacterial product comprising the steps of: providing viable cellsthat express a nuclear hormone receptor and a nuclear hormone substrate,a reporter construct, or both, wherein expression of the substrate, thereporter construct or both is detectable and provides a measurement ofnuclear hormone receptor pathway activity; contacting test cells cellswith a test agent and a bacterial product; contacting control cells witha bacterial product; detecting and comparing nuclear hormone receptorpathway activity between the test and control cells to identify a testagent that inhibits repression of the receptor pathway activity by thebacterial product.
 17. The method of claim 16, wherein the bacterialproduct is anthrax lethal factor (LF) or lethal toxin (LeTx).
 18. Themethod of claim 17, wherein the agent that inhibits or blocks anthraxlethal factor (LF) or lethal toxin (LeTx) repression of nuclear hormonereceptor activity is a cofactor for the nuclear hormone receptor. 19.The method of claim 18, wherein the cofactor is a coactivator for thenuclear hormone receptor.
 20. The method of claim 18, wherein thenuclear hormone receptor is GR.
 21. The method of claim 18, wherein thenuclear hormone receptor is PR.
 22. The method of claim 18, wherein thenuclear hormone receptor is estrogen receptor-α (ER-α).
 23. The methodof claim 18, wherein the effective agent is a genetically engineered orchemically modified variant or mimetic of LF or LeTx, a drug, or acofactor for the nuclear hormone receptor.
 24. The method of claim 24,wherein the effective agent is a co-activator for the nuclear hormonereceptor.
 25. A method for identifying an active protein or othermacromolecule from a cell expressesing a nuclear hormone receptor,wherein the active protein or other macromolecule interacts with abacterial product that modulates nuclear hormone receptor pathwayactivity, comprising the steps of: exposing the bacterial product to alysate or other biological sample from the cell expressing the nuclearhormone receptor under conditions to allow for binding of the bacterialproduct to the active protein or other macromolecule; contacting thebacterial product with a binding partner that provides for isolation oridentification of the bacterial product bound to the active protein orother macromolecule; detecting a bound complex of the bacterial productwith the active protein or other macromolecule; and identifying theactive protein or other macromolecule bound in the complex.
 26. Themethod of claim 25, wherein the binding partner is a polyclonal ormonoclonal antibody that binds the bacterial product.
 27. The method ofclaim 32 which comprises an immunoprecipitation assay.
 28. The method ofclaim 25, wherein the active protein or other macromolecule bound in thecomplex is identified before separation from the complex, or followingan additional step to separate the active protein or other macromoleculefrom the complex.
 29. The method of claim 31, wherein the active proteinor other macromolecule bound in the complex is identified by Westernblotting and/or mass spectroscopy.
 30. A method for alleviating orpreventing one or more symptoms of a bacterial disease, inflammatoryreaction, or autoimmune response in a mammalian subject comprisingadministering an effective amount of an agonist or antagonist of anuclear hormone receptor selected according to the method of claim 1.31. A method for alleviating or preventing one or more symptoms of abacterial disease, inflammatory reaction, or autoimmune response in amammalian subject comprising administering an effective amount of anagent that inhibits or enhances modulation of a nuclear hormone receptorby a bacterial product.
 32. A method for alleviating or preventing oneor more symptoms of anthrax disease and/or an associated inflammatoryreaction, or autoimmnune response, in a mammalian subject comprisingadministering an effective amount of an effective agent that inhibits,blocks, or enhances modulation of activity of one or more nuclearhormone receptor(s) by a anthrax lethal factor (LF) or lethal toxin(LeTx) or an analog, variant, derivative, or mimetic thereof.
 33. Amethod for alleviating or preventing one or more symptoms of a bacterialdisease, inflammatory reaction, or autoimmune response in a mammaliansubject comprising administering an effective amount of a cofactor thatis an agonist or antagonist of a nuclear hormone binding receptor.
 34. Apharmaceutical composition for alleviating or preventing one or moresymptoms of a bacterial disease, inflammatory reaction, or autoimmuneresponse in a mammalian subject comprising an effective amount of anagonist or antagonist of a nuclear hormone receptor selected accordingto the method of claim
 1. 35. A pharmaceutical composition foralleviating or preventing one or more symptoms of a bacterial disease,inflammatory reaction, or autoimmune response in a mammalian subjectcomprising an effective amount of an agent that inhibits or enhancesmodulation of a nuclear hormone receptor by a bacterial product.
 36. Themethod of claim 35, wherein the agent is a cofactor of the nuclearhormone receptor.
 37. A pharmaceutical composition for alleviating orpreventing one or more symptoms of anthrax disease and/or an associatedinflammatory reaction, or autoimmune response, in a mammalian subjectcomprising an effective amount of an effective agent that inhibits,blocks or enhances modulation of activity of one or more nuclear hormonereceptor(s) by an anthrax lethal factor (LF) or lethal toxin (LeTx) oran analog, variant, derivative, or mimetic thereof.
 38. A compositioncomprising a recombinantly or chemically modified analog, fragment orderivative of a bacterial product that exhibits substantially reduced orincreased activity as a modulator of nuclear hormone receptor activitycompared to a native or wild-type counterpart bacterial product.
 39. Thecomposition of claim 38, wherein the composition elicits an immuneresponse against the native or wild-type counterpart bacterial productin a mammalian subject
 40. The immunogenic composition of claim 38,wherein said analog, fragment or derivative comprises a mutant variant,truncated fragment, or chemically modified derivative of an anthraxlethal factor (LF) or lethal toxin (LeTx).
 41. The immunogeniccomposition of claim 40, wherein said LF or LeTx variant, fragment orderivative exhibits substantially reduced or increased activity for GRand/or PR repression.
 42. The immunogenic composition of claim 40,wherein said LF or LeTx variant, fragment or derivative exhibitssubstantial activity as an immunogen, and/or inhibits, blocks, orenhances nuclear hormone repression activity by native LF or LeTx. 43.The immunogenic composition of claim 38, wherein said analog, fragmentor derivative is characterized by a reduction or increase in a level ofnuclear hormone modulation activity of at least 30% compared torepressor modulation activity of a corresponding native bacterialproduct.
 44. A composition comprising a recombinantly or chemicallymodified analog, fragment or derivative of a bacterial product thatinhibits, blocks, or enhances an interaction of a corresponding nativebacterial product with a nuclear hormone receptor.
 45. A method foridentifying an agent of use in treating anthrax, comprising: providingviable cells that express a receptor selected from the group consistingof a glucocorticoid receptor, an estrogen receptor α (ER-α), and aprogresterone receptor B (PR-B) and a nucleic acid comprising aresponsive element selected from the group consisting of aglucocorticoid receptor responsive element, an estrogen receptor α(ER-α) responsive element, and a progresterone receptor B (PR-B)responsive element, respectively, wherein the responsive element isoperably linked to a nucleic acid encoding a polypeptide, whereinexpression of the polypeptide is detectable and provides a measurementof the activity of the glucocorticoid responsive element; contactingtest cells with a test agent and anthrax lethal toxin (LeTx); detectingexpression of the polypeptide, wherein increased expression of thepolypeptide as compared to a control identifies the agent as of use intreating anthrax.
 46. The method of claim 45, wherein the control is atest cell contacted with anthrax lethal toxin in the absence of theagent.
 47. The method of claim 45, wherein the receptor comprises aglucocorticoid receptor and wherein the responsive element is aglucocorticoid responsive element.
 48. The method of claim 45, whereinthe receptor comprises a estrogen receptor α (ER-α) and the responsiveelement comprises a estrogen receptor α (ER-α) responsive element. 49.The method of claim 45, wherein the receptor is a progesterone receptorB (PR-b) and the responsive element comprises a progesterone receptor Bresponsive element.
 50. A agonist of the glucocorticoid receptor, anestrogen receptor α (ER-α), and a progresterone receptor B (PR-B) in themanufacture of a medicament for the treatment of anthrax.
 51. A methodfor treating an anthrax infection, comprising administering to a subjectinfected with anthrax or at risk of infection with anthrax atherapeutically effective amount of an agent that affects the activityof the glucocorticoid receptor, an estrogen receptor a (ER-α), and aprogresterone receptor B (PR-B), thereby treating the anthrax infection.